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Am J Physiol Heart Circ Physiol 277: H1326-H1337, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 4, H1326-H1337, October 1999

Hypoxia stimulates proliferation and interleukin-1alpha production in human vascular smooth muscle cells

Angela L. Cooper and Debbie Beasley

Division of Nephrology, Department of Medicine, New England Medical Center Hospitals and Tufts University School of Medicine, Boston, Massachusetts 02111


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Several lines of evidence indicate that hypoxia is a stimulus to vascular smooth muscle cell (VSMC) proliferation that occurs in pulmonary hypertension. The present study tested the hypothesis that low O2 tension directly stimulates human VSMC proliferation by inducing them to produce interleukin (IL)-1, a potent autocrine growth factor for human VSMC. Human VSMC derived from pulmonary artery, aorta, or saphenous vein were incubated in either a normal in vitro O2 environment (20% O2) or in chambers containing low (~1%) or moderate (5%) O2. Levels of IL-1alpha and IL-1beta mRNA increased in human VSMC after 24-48 h of incubation in low O2 compared with levels in normoxic cells and then decreased upon subsequent reoxygenation. Levels of cell-associated IL-1alpha also increased progressively after 24-48 h in low O2; however, detectable IL-1alpha was not released from the cells in the media. IL-1beta was detectable in cell lysates and supernatants; however, the levels were not affected by exposure to low O2. mRNA encoding for tumor necrosis factor-alpha (TNF-alpha ), a related cytokine and VSMC mitogen, was not detectable in human VSMC exposed to either low or 20% O2. Proliferation of human VSMC was not stimulated during exposure to low O2, despite the fact that cells remained responsive to the mitogenic effect of exogenous IL-1. Interestingly, however, exposure to 5% O2 enhanced proliferation of human VSMC but did not induce IL-1alpha production. Inhibition of IL-1 binding to the type I IL-1 receptor by exogenous addition of IL-1-receptor antagonist (10 µg/ml) did not attenuate the proliferation rates of human VSMC incubated in 20%, 5%, or low O2 or in human VSMC that were reoxygenated after exposure to low O2. These results demonstrate two direct and distinct effects of hypoxia on VSMC. Exposure to moderately low O2 tension induces VSMC proliferation, independent of IL-1, whereas exposure to very low O2 tension induces production of IL-1alpha .

oxygen tension; tumor necrosis factor; interleukin-1-receptor antagonist


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR SMOOTH MUSCLE cells (VSMC) do not proliferate in normal blood vessels from healthy adults. However, excessive VSMC proliferation does occur in vascular diseases, including atherosclerosis and chronic pulmonary hypertension, and it contributes to the pathogenic process in both diseases. A link between hypoxia and smooth muscle cell (SMC) proliferation was first suggested by certain findings in pulmonary hypertension. Chronic hypoxia is often a trigger to pulmonary hypertesion in humans, and the increase in pulmonary vascular resistance is mediated in part via SMC hyperplasia within the media of small pulmonary arteries (27, 50). SMC proliferation has also been demonstrated in the pulmonary vasculature in animal models of chronic hypoxia (44, 62).

More recently, hypoxia has been proposed to play a role in the development of atherosclerotic lesions (15). Cigarette smoking, carbon monoxide exposure, and chronic sleep apnea are associated with hypoxia and increased risk of atherosclerosis (1, 16, 52). Another mechanism that has been proposed to link hypoxia and atherosclerosis involves thrombus formation within the vasa vasorum, leading to hypoperfusion and hypoxia of the adventitia and ultimately inducing the release of cytokines that induce SMC proliferation (43). The cytokines that may contribute to SMC proliferation in this setting have not been identified.

Several in vitro studies have addressed possible mechanisms whereby hypoxia may induce VSMC proliferation. Early studies demonstrated that hypoxic endothelial cells (EC) release a factor(s) that enhances SMC proliferation (56, 57), and subsequent studies have shown that hypoxic EC can release basic fibroblast growth factor and promitogenic prostanoids (45). However, hypoxic EC can also release potential inhibitors of VSMC proliferation (29, 30); thus, the overall effect of EC-derived factors on SMC mitogenesis during hypoxia is unclear. Exposure to hypoxia can also stimulate proliferation of bovine VSMC directly. Bovine SMC proliferate faster when exposed to moderate hypoxia (3% O2) but only if the cells are costimulated with phorbol ester (18, 17) or serum (19). These results suggest that hypoxia provides a comitogenic stimulus in the presence of other growth factors. However, it is not known whether hypoxia has either mitogenic or comitogenic effects on human VSMC or whether hypoxia induces human VSMC to produce an autocrine growth factor.

The proinflammatory cytokine interleukin (IL)-1 is an intriguing candidate for a hypoxia-induced SMC-derived autocrine growth factor. Human VSMC produce both the alpha  and beta  forms of IL-1 (6, 39), and both IL-1alpha and IL-1beta are potent VSMC growth factors (40, 4). IL-1alpha is also a potent autocrine growth factor, as indicated by the markedly enhanced proliferative rate of human VSMC that have been stably transfected with IL-1alpha expression plasmids that direct the production of low (pg) levels of IL-1alpha (4). Finally, hypoxia can induce IL-1 production, as demonstrated by in vitro studies in which exposure to severe hypoxia (0% O2) induces the release of bioactive IL-1 from human EC (54). However, the effect of hypoxia on IL-1 production may be cell type specific, since human macrophages, unlike EC, do not produce IL-1 when exposed to hypoxia but produce it upon subsequent reoxygenation (24, 35).

The present studies first assessed whether exposure to severe hypoxia, or alternatively reoxygenation after hypoxia, induces IL-1 production by human VSMC. Severe hypoxia itself stimulated IL-1alpha production in human VSMC, reoxygenation reversed this effect, and the levels of IL-1alpha produced were similar to those that stimulate proliferation in human VSMC that have been stably transfected with IL-1alpha expression plasmids. Therefore, further studies were designed to test the hypothesis that either moderate or severe hypoxia stimulates proliferation of human VSMC by inducing autocrine production of IL-1alpha .


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Human VSMC culture. Segments of pulmonary artery, aorta, and saphenous vein were obtained from organ donors (National Disease Research Interchange, Philadelphia, PA). Segments of saphenous vein were also obtained from coronary bypass patients at New England Medical Center (approved by the Human Investigation Research Committee). Human VSMC were cultured from explants as previously described (5). Cells were grown in DMEM supplemented with 10% FCS, penicillin, streptomycin, and fungizone and were passaged every 2-4 wk using trypsin-EDTA. Cells were passaged at a 1:3 ratio and were used after reaching confluence, at passage 1-4. In some experiments, human VSMC were serum deprived in DMEM-Ham's F-12 media (1:1) supplemented with insulin (1 µM) and transferrin (5 µg/ml; DMEM-F-12-IT) for 48 h before exposure to hypoxia, and then the medium was changed to fresh DMEM-F-12-IT. In other experiments, human VSMC were exposed to hypoxia in DMEM supplemented with 1-10% FCS.

Exposure of human VSMC to hypoxia/reoxygenation. VSMC were exposed to low O2 tension in a humidified modular incubator chamber (Billups-Rothenberg, Del Mar, CA) that was maintained at 37°C. To expose VSMC to low O2, the incubation medium was pregassed for 1 h with 95% N2-5% CO2 before the experiment. VSMC were washed two times with PBS, the medium was replaced with pregassed low O2 medium, and the culture plates placed in the incubator chamber, which was sealed and purged with 95% N2-5% CO2 for 20 min. Chambers were reequilibrated with 95% N2-5% CO2 at 24-h intervals. Reoxygenation involved transferring the cells to 95% ambient air-5% CO2 at 37°C. VSMC were exposed to intermediate (5%) O2 levels as described above, except the medium was not pregassed, and the incubator chamber was purged with 90% N2-5% O2-5% CO2 for 20 min. Normoxic control VSMC were exposed to 95% ambient air-5% CO2 for the entire incubation period.

A portable O2 analyzer (Hudson, Ventronics Division) was used to determine the O2 content of the air inside the chamber at the end of the hypoxic exposure period. When chambers were purged with 0% O2, O2 readings were either 0 or 1% (to the nearest percentage) at the end of the hypoxia period (referred to as low O2 in text). O2 readings were consistently 5% when chambers were purged with 5% O2. One experiment was excluded from analysis, since low O2 was not sustained at the end of the incubation period. Hypoxia had no effect on the pH of the medium. The viability of human pulmonary artery SMC (HPASMC), human aortic SMC (HASMC), and human saphenous vein SMC (HSVSMC), assessed by trypan blue exclusion and by measuring lactate dehydrogenase activity in cell supernatants (Promega, Madison, WI), was not affected by incubation in low O2, and cell morphology was unchanged.

RT-PCR. Total RNA was extracted from human VSMC using RNAzol B (Biotecx Laboratories, Houston, TX). RNA was reverse transcribed for PCR analysis in a total volume of 20 µl containing 2 µg RNA, 200 units of Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL), 25 µg/ml oligo(dT) primer (GIBCO-BRL), 0.5 mM dNTP, and 10 mM dithiothreitol in the manufacturer's recommended buffer. The reactions were carried out at 37°C for 1 h and were terminated by heating to 95°C for 5 min. PCR mixtures contained 2 µl of cDNA, 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 1.0-1.5 mM MgCl2, 0.01 mg/ml gelatin, 200 µM of each deoxynucleotide phosphate, 0.15 µCi [32P]dCTP (20 nM), 250 nM of each primer, and 1.25 units of Taq polymerase (Pharmacia) in a final volume of 20 µl. Primer sequences and annealing temperatures are given in Table 1. Amplification reactions were begun with an initial melt of 94°C for 2 min, consisted of 16-32 cycles of 1 min at 94°C, 1 min at the annealing temperature, and 2 min at 72°, and were completed with a final extension at 72°C for 5 min. Each sample was analyzed in two or three reactions of differing cycle number, within the exponential phase of amplification. Products were separated by gel electrophoresis on a 5% polyacrylamide gel and were visualized by ethidium bromide staining. PCR products were quantitated by cutting the band from the gel and counting the incorporated [32P]dCTP by liquid scintillation counting. Levels of mRNA were determined by normalizing the amount of each PCR product (counts/min) to the amount of beta -actin product obtained for the same cDNA sample. The level of mRNA in cells exposed to hypoxia was expressed relative to the value for VSMC exposed to 20% O2 alone for the same time period.

                              
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  		Table 1.   Primer sequences and PCR product size

IL-1alpha , IL-1beta , and IL-1-receptor antagonist measurements. Cell lysates were prepared by three successive freeze-thaw cycles in buffer containing 10 mM sodium phosphate, 0.15 M NaCl, 0.25% BSA, and 0.05% sodium azide. Both lysates and supernatants were cleared of cellular debris by centrifugation at 500 g for 10 min. Immunoreactive IL-1alpha in cell lysates and cell supernatants was measured by an enzyme immunometric assay (EIA) that detects both IL-1alpha precursor and mature IL-1alpha (Cayman Chemical, Ann Arbor, MI) at a detection limit of 1-2 pg/ml (26). IL-1beta in cell supernatants was measured by an EIA (Cayman Chemical) that detects mature IL-1beta (the active form of IL-1beta ) preferentially, at a detection limit of 1-2 pg/ml. Immunoreactive IL-1-receptor antagonist (ra) in cell lystes was measured by a specific RIA that detects both secreted and intracellular (ic) forms of IL-1ra with a detection limit of 35-70 pg/ml (49).

Bromodeoxyuridine incorporation. Human VSMC were plated at 2,000 cells/well in 96-well plates in DMEM supplemented with 10% FCS. After the cells were attached to the plastic, the medium was replaced with DMEM supplemented with 2% FCS, and the plates were transferred to low, 5%, or 20% O2. After 3 days, the medium was changed to fresh DMEM containing 2% FCS and 5-bromo-2'-deoxyuridine (BrdU; Boehringer Mannheim). After 24 h, the cells were fixed, and DNA was denatured; next, the fixed cells were incubated 2 h at room temperature with mouse monoclonal BrdU antibody conjugated to peroxidase (Boehringer Mannheim). Tetramethylbenzidine was used as substrate, and absorbance at 405 nm was determined. In some of the experiments, human recombinant IL-1ra (10 µg/ml) or IL-1beta (1 ng/ml) was added to the media before exposure to hypoxia or normoxia and was also added to the fresh media containing BrdU.

Proliferation rates. Human VSMC were plated in 24-well plates at a density of 5,000 cells/cm2, in complete growth media containing 10% FCS. After the cells had adhered to the plastic, the medium was replaced with media supplemented with 1, 2, 5, or 10% FCS, and the plates were transferred to low, 5%, or 20% O2. Fresh medium was replaced every 3-4 days. Quadruplicate wells of cells exposed to each O2 concentration were trypsinized on the day after plating (day 0) and after 4-13 days of exposure to low O2, and cell number was determined using a Coulter Counter.

Data analysis. Values presented are means ± SE of four to eight replicate cultures for each treatment group in a representative experiment. Values that are below the limit of detection of the EIA are indicated by ND (nondetectable). Within experiments, the significance of treatment-induced differences was determined either by t-test or by ANOVA followed by Dunnett's procedure to compare multiple means with a single control value. P < 0.05 was considered statistically significant. Each experiment was repeated in VSMC derived from different blood vessels and different patients or donors, as described.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IL-1alpha and IL-1beta gene expression are enhanced in human VSMC incubated in low O2. Because the pulmonary vasculature is sensitive to the effects of hypoxia in vivo, the effects of low (~1%) O2 and subsequent reoxygenation on cytokine gene expression were initially assessed in SMC derived from HPASMC. Both IL-1alpha and IL-1beta mRNA were detectable in HPASMC before exposure to low O2. After exposure to low O2 tension for 48 h, IL-1alpha mRNA levels increased 3.7-fold compared with the levels in HPASMC incubated in 20% O2, and IL-1beta mRNA levels increased 1.7-fold (Fig. 1). Subsequent reoxygenation rapidly reversed the effects of low O2 on IL-1alpha and IL-1beta mRNA. Both IL-1alpha and IL-1beta mRNA levels remained moderately elevated after 4 h of reoxygenation (1.9- and 1.4-fold, respectively) but were not different from time controls after 8 h of reoxygenation. Levels of beta -actin mRNA were similar in HPASMC incubated in either 20% O2 or low O2 and were not affected by reoxygenation.


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  		Fig. 1.   Exposure to low O2 increases levels of interleukin (IL)-1alpha , IL-1beta , and intracellular (ic) IL-1-receptor antagonist (ra) mRNA in human pulmonary artery smooth muscle cells (HPASMC). RNA was isolated from HPASMC after 48 h of exposure to low O2 alone (lane 2) or after 4 or 8 h of reoxygenation (lanes 4 and 6, respectively). Levels of mRNA were analyzed by RT-PCR with amplification within the exponential phase [cycles of PCR amplification: IL-1alpha , (28); IL-1beta (28); tumor necrosis factor-alpha (TNF-alpha ; see Ref. 30), icIL-1ra (32); and beta -actin (18)]. Corresponding time controls (48, 52, or 56 h in 20% O2) are shown in lanes 1, 3, and 5, respectively. RT-PCR products obtained from HSVSMC treated with IL-1beta (2 ng/ml) for 24 h are shown in lane 7.

Tumor necrosis factor-alpha (TNF-alpha ) mRNA was not detectable in either control HPASMC or HPASMC that had been exposed to low O2 tension for 48 h. PCR product was not visible on the ethidium bromide-stained gel (Fig. 1) and was not detectable as incorporated [32P]dCTP, even after 32 cycles of amplification. In contrast, RNA prepared from HSVSMC stimulated with IL-1beta (2 ng/ml) for 24 h gave an intense PCR product of the expected size after 30 cycles of amplification, verifying the sensitivity of the RT-PCR method for detection of TNF-alpha mRNA.

SMC derived from other blood vessels were studied to assess whether the effects of hypoxia on IL-1alpha are specific to HPASMC. Exposure to low O2 tension for 48 h induced similar increases in IL-1alpha and IL-1beta mRNA in HASMC. Levels of IL-1alpha mRNA increased threefold compared with time controls incubated in normal O2 tension, whereas levels of IL-1beta mRNA increased 2.2-fold (data not shown).

Cell-associated IL-1alpha is increased in human VSMC incubated in low O2. Incubation in a low-O2 environment stimulated IL-1alpha production by human VSMC. Cell-associated IL-1alpha levels increased progressively in HPASMC exposed to low O2 for 12-48 h (Fig. 2A). In contrast, the level of cell-associated IL-1alpha in control HPASMC decreased throughout the course of the experiment, an effect that may be due to progressive serum deprivation (48 h serum deprivation at the start of the experiment vs. 96 h by the end of the experiment). When compared with the corresponding time control HPASMC, cell-associated IL-1alpha levels were increased in hypoxic HPASMC by 1.5-, 2.5-, and 18-fold, respectively, after 12, 24, and 48 h of exposure to low O2.


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  		Fig. 2.   A: time-dependent increase in cell-associated IL-1alpha in HPASMC exposed to low O2 for 4-48 h. B: cell-associated IL-1alpha levels decreased in HPASMC that were reoxygenated after 48 h of exposure to low O2 and were nondetectable in the corresponding time controls. HPASMC were serum deprived for 48 h before exposure to low O2 in fresh serum-free media. At the times indicated, cells were lysed in enzyme immunometric assay (EIA) buffer, and cellular levels of IL-1alpha were determined by EIA. ND, nondetectable (<0.04 ng/106 cells). IL-1alpha was not detectable in cell supernatants. * P < 0.002, significantly different from 20% O2 at same time point.

Subsequent reoxygenation of HPASMC exposed to low O2 caused a decline in cell-associated IL-1alpha levels. In the experiment shown in Fig. 2B, cell-associated IL-1alpha increased 350-fold in HPASMC incubated 48 h in low O2. Cell-associated IL-1alpha began to decrease after only 4 h of reoxygenation but remained significantly elevated compared with HPASMC incubated in normal O2 after reoxygenation for 24 h. The reversal of IL-1alpha levels upon reoxygenation provides further evidence that IL-1alpha production was not due to a nonspecific toxic effect of O2 deprivation.

Exposure to low O2 also induced IL-1alpha production in HASMC (Fig. 3A). Cell-associated levels of IL-1alpha increased fourfold in HASMC exposed to low O2 for 48 h. Subsequent reoxygenation for 9 h reduced IL-1alpha levels in the lysates of hypoxic HASMC; however, IL-1alpha levels remained significantly greater than the corresponding time controls. Similar results were obtained with four different lines of saphenous vein SMC that were derived from four different coronary bypass patients (Fig. 3B). The effect of low O2 on IL-1alpha production was similar when cells were incubated either in the presence or absence of serum (Table 2). Cell-associated IL-1alpha was not detectable in HSVSMC incubated in normal O2 in the absence of FCS but was detectable in HSVSMC incubated in normal O2 in the presence of 10% FCS (0.05 ± 0.02 ng/106 cells), indicating that FCS is a weak inducer of IL-1alpha production. After exposure to low O2 for 48 h, cell-associated IL-1alpha increased to similar levels in cells incubated without FCS or with 10% FCS (2.40 ± 0.3 and 2.56 ± 0.94 ng/106 cells, respectively).


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  		Fig. 3.   Exposure to low O2 increases cell-associated IL-1alpha in VSMC derived from human aorta (HASMC) or saphenous vein (HSVSMC). A: HASMC were exposed to low or 20% O2 for 48 h, followed by 0 or 9 h of reoxygenation or continued oxygenation, as in Fig. 2B, except serum deprivation was begun upon exposure to low O2. B: HSVSMC (passage 2) derived from 4 different patients were exposed to 20% or low O2 for 48 h, as in Fig. 2A. ND, <0.02 ng/106 cells. * P < 0.01, significantly different from 20% O2 at same time point.


                              
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  		Table 2.   Effect of low O2 on IL-1alpha , IL-1beta , and IL-1ra

In contrast to the stimulatory effect of low O2 on IL-1alpha production, low O2 did not stimulate IL-1alpha release by human VSMC. IL-1alpha was not detectable (<2 pg/ml) in 48-h supernatants of human VSMC incubated in either low or 20% O2 (n = 10 experiments with HPASMC, HSVSMC, and HASMC, including experiments in Figs. 2A, 3A, and 3B and 4 experiments not shown). These results indicate that hypoxic human VSMC do not effectively release IL-1alpha . Reoxygenation after hypoxia was also not an effective stimulus for IL-1alpha release. IL-1alpha was not detectable (<2 pg/ml) in supernatants of human VSMC that were reoxygenated for 9 h after the 48-h exposure to low O2 (n = 4 experiments with HPASMC, HSVSMC, and HASMC). In one experiment, IL-1alpha was detectable in the supernatant of reoxygenated HPASMC; however, the levels were low (2.7 ± 0.3 pg/ml compared with <2 pg/ml for time control cells). Thus cellular release of IL-1alpha is unlikely to account for the decline in cell-associated IL-1alpha that occurs during reoxygenation.

Exposure to low O2 did not stimulate IL-1beta release by human VSMC. In five experiments (3 with HSVSMC and 2 with HPASMC), supernatants obtained from cells after 48 h of exposure to low or 20% O2 contained detectable IL-1beta ; however, there was no effect of O2 tension on the amount of IL-1beta detected (Table 2). Cell-associated IL-1beta was also measured in one experiment and was likewise detectable and not significantly different between HPASMC incubated in low versus 20% O2 (data not shown).

Proliferation of human VSMC in low O2 is not influenced by IL-1 receptor activation. To test whether IL-1alpha produced during exposure to low O2 had a proliferative effect on human VSMC, BrdU incorporation was measured in cells that had been exposed to low O2 for 72-96 h to allow sufficient time for induction of IL-1alpha synthesis and for the produced IL-1alpha to enhance DNA synthesis. Exposure to low O2 did not significantly affect the proliferative rate of human VSMC. In seven experiments with different human VSMC cell lines (HPASMC, HASMC, and HSVSMC), BrdU incorporation was not significantly enhanced by exposure to low O2 in the presence of 2% FCS (1.08 ± 0.24 relative to normal O2). Although there was a tendency for BrdU incorporation to be increased in passage 1 human VSMC exposed to low O2 (1.54 ± 0.42 relative to 20% O2, n = 3 experiments) and attenuated in passage 3-4 human VSMC that were exposed to low O2 (0.74 ± 0.12 relative to normal O2, n = 4 experiments), neither of these effects was statistically significant. A representative experiment with passage 1 HSVSMC is shown in Fig. 4.


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  		Fig. 4.   Exposure to 5% O2, but not low O2, stimulates DNA synthesis in HSVSMC; proliferation is not affected by exogenous IL-1ra. HSVSMC (passage 1) were plated in 96-well plates in complete media. After cells had attached to the plastic, medium was changed to DMEM supplemented with 2% FCS, and some of the plates were transferred to either 5% or low O2 for 72 h. At 72 h, the medium was replaced with fresh DMEM supplemented with 2% FCS and bromodeoxyuridine (BrdU). Some of the cells were exposed to IL-1ra (10 µg/ml) from the time of plating, with fresh IL-1ra added with the BrdU-containing media. BrdU incorporation was determined at 96 h. * P < 0.002, significantly different from 20% O2 without IL-1ra.

It is possible that IL-1alpha produced by VSMC exposed to low O2 promotes VSMC proliferation, but the pro-proliferative effect of IL-1alpha is counteracted by hypoxia-induced antiproliferative effects. To test whether proliferation of human VSMC in low O2 was affected by IL-1 receptor signaling, human VSMC were exposed to IL-1ra before exposure to hypoxia to inhibit binding of IL-1alpha (or IL-1beta ) to the type I IL-1 signaling receptor (28). IL-1ra (10 µg/ml) was added before the 72-h incubation in 20% or low O2 and was readded with the BrdU labeling media. Exogenous IL-1ra did not affect BrdU incorporation in human VSMC, including HPASMC, HASMC, and HSVSMC, which were incubated in 20% O2 (0.96 ± 0.04 relative to without IL-1ra, n = 8 experiments), or in human VSMC that were incubated in low O2 (0.94 ± 0.06 relative to without IL-1ra, n = 6 experiments). A representative experiment with passage 1 HSVSMC is shown in Fig. 4. Thus IL-1ra failed to identify an IL-1 receptor-dependent component of proliferation in a low-O2 environment.

It is possible that human VSMC incubated in low O2 are insensitive to the proproliferative effects of IL-1. Human EC produce and release IL-1 when exposed to very low O2, but the released IL-1 is not effective. Upon reoxygenation, however, human EC display IL-1-induced expression of adhesion molecules (54). To assess whether IL-1 can be an effective mitogen in the presence of low O2, the proliferative effects of exogenous IL-1 were compared in human VSMC exposed to low O2 vs. 20% O2. Exogenous IL-1beta (1 ng/ml) increased BrdU incorporation 85% in HSVSMC incubated in 20% O2 and 94% in the presence of low O2 (Fig. 5). In contrast, the proliferative response to 10% FCS was attenuated in low O2 compared with 20% O2 (217 and 122%, respectively). Similar results were obtained with human VSMC derived from another patient.


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  		Fig. 5.   Exposure to low O2 does not attenuate the mitogenic response to exogenous IL-1 in human VSMC. Human VSMC were plated as described in Fig. 4, and medium was changed to DMEM supplemented with either 2% FCS, 2% FCS and IL-1beta (1 ng/ml), or 10% FCS. After 72 h, medium was replaced with fresh medium containing BrdU, and BrdU incorporation was determined at 96 h. BrdU incorporation is expressed relative to values for cells incubated in the presence of 2% FCS and 20% O2. * P < 0.0005, significantly different from control at the same O2 level. + P < 0.0001, significantly different from 10% FCS and 20% O2.

Because cell-associated IL-1alpha levels remained elevated during the first 24 h of reoxygenation (Fig. 2B), the possibility that subsequent reoxygenation induces proliferation of human VSMC via IL-1-dependent mechanisms was also assessed. Proliferation was enhanced more than twofold in human VSMC that were returned to 20% O2 after a 72-h exposure to low O2 (n = 4 experiments, Fig. 6). Exogenous IL-1ra (10 µg/ml) did not attenuate BrdU incorporation in reoxygenated human VSMC, indicating that reoxygenation-induced proliferation is also independent of IL-1 receptor activation.


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  		Fig. 6.   Reoxgenation after exposure to low O2 significantly enhances proliferation of human VSMC; proliferation is not affected by exogenous IL-1ra. Human VSMC were plated as described in Fig. 4, and medium was changed to DMEM supplemented with 2% FCS with or without IL-1ra (10 µg/ml). Some of the plates were transferred to low O2 for 72 h, and then medium was replaced with fresh DMEM supplemented with 2% FCS and BrdU, with or without IL-1ra, and all plates were returned to 20% O2. BrdU incorporation was determined at 96 h. Values shown are means ± SE of 4 experiments with HSVSMC, HASMC, and HPASMC. BrdU incorporation is expressed relative to values for cells incubated in 20% O2 without IL-1ra. * P < 0.02, significantly different from 20% O2 without IL-1ra.

Exposure to low O2 induces modest increases in production of icIL-1ra. Previous studies have shown that HSVSMC produce icIL-1ra, a potential inhibitor of IL-1 action, and its production is stimulated by various stimuli, including IL-1 and platelet-derived growth factor (6). Expression of icIL-1ra mRNA was enhanced in HPASMC after exposure to low O2 tension for 48 h. cDNA prepared from HPASMC that had been exposed to low O2 tension for 48 h yielded visible PCR product of the expected size (510 bp) after 32 cycles of amplification, whereas cDNA prepared from HPASMC incubated in 20% O2 did not yield visible product (Fig. 1). However, the magnitude of the hypoxia-induced increase in icIL-1ra was modest compared with the magnitude of the induction elicited by IL-1beta (Fig. 1), a known potent activator of icIL-1ra production (6). The level of icIL-1ra transcript was 10-fold lower in HPASMC exposed to low O2 compared with HSVSMC stimulated with IL-1beta (2 ng/ml) for 24 h, as determined by quantitation of the PCR bands.

Exposure to low O2 also induced detectable production of IL-1ra in human VSMC but only in cells that were costimulated with serum (Table 2). Cell-associated IL-1ra was not detectable in human VSMC exposed to either 20% or low O2 for 48 h in the absence of serum (n = 6 experiments, 4 with HSVSMC, 1 with HPASMC, and 1 with HASMC). In the presence of 10% FCS, cell-associated IL-1ra was also not detectable in the cell lysates of HSVSMC incubated in 20% O2 but was detectable in HSVSMC incubated in low O2 (1.00 ± 0.33 ng/106 cells). FCS alone is likely a weak stimulus to IL-1ra production, since platelet-derived growth factor induces modest increases in IL-1ra production (6). Hypoxia may also be a weak stimulus to IL-1ra production, since a modest increase in icIL-1 mRNA was observed when HSVSMC were exposed to low O2 in the absence of serum (Fig. 1). IL-1ra levels may be below the detection limit of the assay (<0.3 ng/106 cells) in HSVSMC stimulated with hypoxia or FCS alone, whereas both stimuli together significantly augment IL-1ra production.

Exposure to 5% O2 enhances human VSMC proliferation by an IL-1-independent mechanism. Exposure to 5% O2 tension consistently enhanced BrdU incorporation in human VSMC, including HPASMC, HASMC, and HSVSMC. The effect of 5% O2 was similar in SMC derived from different blood vessels. BrdU incorporation was consistently increased after 72 h of exposure to 5% O2 in the presence of 2% FCS (1.55 ± 0.11 relative to 20% O2, P < 0.002, n = 9 experiments). Passage 1 human VSMC responded more to the effect of 5% O2; BrdU incorporation increased by 1.76 ± 0.13 relative to 20% O2 (n = 5 experiments, P < 0.005). A representative experiment with passage 1 HSVSMC is shown in Fig. 4. BrdU incorporation was also enhanced in passage 3 or 4 human VSMC incubated in 5% O2; however, the magnitude of the effect was less (1.28 ± 0.03 relative to 20% O2, n = 4, P < 0.003).

Direct cell counting confirmed that exposure to a 5% O2 environment for four or more days enhances the proliferative rate of human VSMC. The increment in cell counts obtained after incubation for 4-13 days in either 5% or 20% O2 was compared in five experiments with passage 1 HPASMC and HSVSMC. Lowering ambient O2 to 5% increased proliferation in the presence of 10% FCS (1.76 ± 0.23 relative to 20% O2, n = 5 experiments, P < 0.03). The proliferative effect of 5% O2 was more marked in the presence of low concentrations of FCS; a representative experiment is shown in Fig. 7A. HSVSMC incubated 13 days in the presence of 1% FCS did not proliferate in a 20% O2 environment but did proliferate in 5% O2. When incubated in media supplemented with 2 or 5% FCS, proliferation rates in 5% O2 were increased 4.5- and 2.5-fold, respectively, relative to 20% O2. Similar results were obtained with HSVSMC derived from another patient (data not shown) and with HPASMC that were exposed to 5% O2 for a shorter period (Fig. 7B). HPASMC incubated 4 days in media supplemented with 2% FCS did not proliferate when incubated in 20% O2 but did when incubated in 5% O2. In the presence of 5 or 10% FCS, proliferation rates were increased 3.7- and 2.1-fold, respectively, relative to 20% O2.


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  		Fig. 7.   Exposure to 5% O2 stimulates proliferation of HPASMC and HSVSMC in media containing either low or high concentrations of FCS. A: HSVSMC (passage 1) were plated in DMEM supplemented with 10% FCS overnight. Initial cell counts were determined by Coulter counter (day 0 = 2,330 cells/cm2), and the medium was changed on the other 2 plates to DMEM supplemented with varying concentrations of FCS. One of the plates was then transferred to 5% O2 for 13 days. Fresh medium was replaced every 3-4 days. Increments in cell counts (day 13 - day 0) are shown. B: HPASMC (passage 1) were studied as in A, except the cells were incubated 4 days in 5% O2 with no media changes. Increments in cell counts (day 4 - day 0) are shown; cell counts on day 0 = 5,060 cells/cm2. * P < 0.001 compared with human VSMC incubated in 20% O2 and the same concentration of FCS.

In distinct contrast to the stimulatory effect of low O2, IL-1alpha production was not stimulated by lowering O2 from 20 to 5%. Cell-associated IL-1alpha was measured in six experiments in which either BrdU incorporation or cell proliferation was significantly enhanced by 5% O2 (3 with HPASMC, 1 with HASMC, and 2 with HSVSMC). In all experiments, cell-associated IL-1alpha was not detectable in cells incubated 4-8 days in the presence of 2% FCS and either 20 or 5% O2 (Table 3). In addition, HASMC or HSVSMC incubated 3 days in 5 or 20% O2 did not release detectable IL-1beta during the subsequent 24-h period when BrdU incorporation was measured (Table 3). Cell-associated IL-1alpha was detectable in human VSMC cultures incubated 4-8 days in normal O2 tension and the presence of 10% FCS (0.19 ± 0.11 ng/106 cells; Table 3). However, the level of cell-associated IL-1alpha was not significantly affected by incubation in 5 versus 20% O2 (n = 6 experiments with HPASMC and with HSVSMC, P = 0.29).

                              
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  		Table 3.   Effect of 5% O2 on IL-1alpha and IL-1beta

It is possible that lowering O2 to 5% induces production of IL-1alpha , but the IL-1alpha produced is not present in cell lysates in a form that is detectable by the IL-1alpha sandwich EIA that was used. For example, membrane-associated IL-1alpha may not be detected because a positive signal in this assay requires two distinct epitopes on the IL-1alpha molecule to be available for binding to the capture and detection antibodies. However, studies with exogenous addition of IL-1ra further rule out the possibility that IL-1 receptor activation contributes to the pro-proliferative effect of 5% O2. IL-1ra (10 µg/ml) did not affect the rate of BrdU incorporation in human VSMC incubated 72-96 h in 5% O2 (1.01 ± 0.07 relative to the absence of IL-1ra, n = 8 experiments). A representative experiment with passage 1 HSVSMC is shown in Fig. 4.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that exposure to very low O2 levels induces IL-1alpha gene expression and increases the cell-associated levels of IL-1alpha in VSMC derived from human aorta, pulmonary artery, and saphenous vein. Cell-associated levels of IL-1alpha increased progressively after exposure to low O2 and returned to control levels upon subsequent reoxygenation. IL-1alpha levels were not increased in human VSMC incubated in 5% O2, indicating that a severe hypoxic stimulus is required to stimulate IL-1alpha production. IL-1alpha began to increase after 12 h of exposure to 0% O2 and markedly increased after 48 h. In contrast, human EC produce IL-1 after only 4 h of exposure to 0% O2 (54), and human mononuclear phagocytes produce IL-1 when reoxygenated after only 9 h of exposure to 0% O2. Shorter periods of low O2 tension appear to induce IL-1alpha production by EC compared with SMC. It is possible that 0% O2 represents a stronger hypoxic stimulus for EC, since they are exposed to a higher range of O2 tension in vivo compared with SMC. In vivo, EC are exposed directly to O2 tensions that occur in blood. In contrast, VSMC can be relatively distant from the blood, depending on the thickness of the blood vessel, their location in the vessel wall, and the presence or absence of vasa vasorum, and thus can be exposed to O2 tensions that are significantly lower than that of blood.

Evidence from in vitro studies suggests that IL-1 produced by vascular cells versus mononuclear phagocytes may be produced by distinct stimuli and may have distinct roles in vivo. Monocytes do not produce IL-1 during hypoxia; however, newly synthesized IL-1 is released during the subsequent reoxygenation period, and the stimulus for IL-1 synthesis is the generation of O2 free radicals (35). Because IL-1 is produced during exposure to low O2 tension in both EC (54) and VSMC, the stimulus to induction of IL-1alpha may not be generation of O2 free radicals. VSMC-derived IL-1 may play a role in pathophysiology associated with chronic ischemia or hypoxia, whereas mononuclear phagocyte-derived IL-1 may contribute to reperfusion injury.

Hypoxia also induced a modest increase in the levels of IL-1beta mRNA in HPASMC. IL-1beta , like IL-1alpha , is initially synthesized as a precursor protein that is cleaved to the mature form by IL-1beta -converting enzyme and subsequently released. However, in contrast to IL-1alpha , the precursor form of IL-1beta is inactive (46); IL-1beta is only active when released as a mature molecule that activates IL-1 receptors. In the present study, lowering O2 tension from 20% to either 5 or ~1% did not enhance IL-1beta release from human VSMC. Thus IL-1beta is unlikely to have autocrine effects in hypoxic human VSMC. Likewise, TNF-alpha , which is also produced by human VSMC (58) and induces VSMC proliferation (48, 51), is unlikely to mediate the effects of hypoxia or reoxygenation in cultured human VSMC, since TNF-alpha mRNA was not expressed by HPASMC that were exposed to low O2 or subsequent reoxygenation.

IL-1alpha produced by human VSMC that were exposed to low O2 remained cell associated and was not released from the cells either during the hypoxia or reoxygenation periods. Human EC also produce IL-1alpha when exposed to 0% O2; however, unlike VSMC, IL-1alpha is released from hypoxic EC into the supernatant (54). When stimulated with IL-1, TNF, or lipopolysaccharide, human EC likewise release IL-1, whereas human VSMC produce IL-1alpha but do not release it (6, 41). Together these findings suggest that IL-1alpha may be more efficiently released by human EC compared with human VSMC. It is not entirely clear whether cellular release of IL-1alpha is required for its subsequent bioactivity, since IL-1alpha has been proposed to affect cellular function by alternative pathways. The precursor form of IL-1alpha is thought to associate with the plasma membrane of human VSMC and macrophages in a form that can stimulate IL-1 receptors on cells that directly contact IL-1alpha -producing cells (13, 38, 41, 63). IL-1alpha precursor may also affect gene expression by localizing directly to the nucleus, without prior release from the cell. IL-1alpha precursor contains a nuclear localization sequence in the precursor domain of the molecule and localizes to the nucleus of EC, fibroblasts, and VSMC that have been transiently transfected with IL-1alpha precursor expression plasmids (4, 42, 60). Also, stable transfection of human EC and VSMC with IL-1alpha precursor expression plasmids produces effects similar to exogenous IL-1; however, these effects are not reversed by exogenous IL-1ra in human EC (42) and are only partially reversed in human VSMC (4). Therefore, it is possible that IL-1alpha produced by hypoxic human VSMC is biologically active in the absence of cellular release.

Previous studies have documented that IL-1 is a potent growth factor for VSMC, including those subcultured from human pulmonary artery or saphenous vein (40, 4) and primary cultures of rat aortic SMC (RASMC; see Ref. 10). However, IL-1 can also induce growth-inhibitory pathways, and the overall effect on proliferation is highly dependent on the type of VSMC tested, the specific assay conditions used, and the length of IL-1 exposure. IL-1 can inhibit proliferation of subcultured RASMC by inducing the expression of inducible nitric oxide (NO) synthase and the generation of growth-inhibitory NO (14, 23, 53). Human VSMC do not express inducible NO synthase when stimulated with IL-1 (5) and thus provide a more suitable model for studies of the pro-proliferative actions of IL-1. IL-1 can also induce the production of growth-inhibitory prostanoids, which suppress the early mitogenic response; IL-1 induces DNA synthesis only in SMC treated with indomethacin when assessed 24-48 h after initial exposure (40, 36). In the present study, exogenous IL-1 was an effective mitogen in the absence of indomethacin when assessed after longer exposures to IL-1 (72-96 h), consistent with earlier findings with RASMC (31). Also, inhibition of prostanoid synthesis with indomethacin did not significantly affect the proliferative responses produced by either IL-1 or low O2 (data not shown).

Studies with transfected human VSMC have shown that IL-1alpha is also a potent autocrine growth factor for these cells. Human VSMC that have been stably transfected with IL-1alpha expression plasmids proliferate rapidly compared with human VSMC transfected with vector alone (4). Exposure to low O2 induces human VSMC to produce IL-1alpha at levels similar to or greater than the levels produced by IL-1alpha stable transfectants. In contrast with IL-1alpha stable transfectants which proliferate rapidly, proliferation was not significantly affected in human VSMC exposed to low O2. It is not clear why proliferation is not enhanced in hypoxic human VSMC despite the fact that they are producing IL-1alpha similar to that of IL-1alpha stable transfectants, which proliferate rapidly. Human EC exposed to severe hypoxia released IL-1 but were insensitive to its effects, and then, upon reoxygenation, the cells continued to release IL-1 and displayed IL-1-dependent expression of leukocyte adhesion (12, 54). In contrast, human VSMC remained responsive to the proliferative effects of IL-1 when incubated in low O2, suggesting that insensitivity to released IL-1 does not account for the lack of IL-1-dependent proliferation in human VSMC exposed to low O2. However, we cannot rule out the possibility that hypoxic VSMC are insensitive to nuclear or membrane-associated IL-1alpha or that IL-1alpha is not effectively targeted to its active site in hypoxic VSMC. Alternatively, it is possible that IL-1alpha is only active when released by VSMC. Stable transfectants release low levels of IL-1alpha (pg/ml), and these low levels can induce human VSMC proliferation (4). In contrast, hypoxic VSMC do not release IL-1alpha either during exposure to hypoxia or during the subsequent reoxygenation period, and proliferation during either hypoxia or reoxygenation is not dependent on IL-1 receptor activation. Interestingly, human VSMC, including those derived from human pulmonary artery, aorta, and saphenous vein, proliferated faster in a 5% O2 environment compared with in 20% O2 when assessed in the presence of either low or high serum concentrations. The finding that serum-induced VSMC proliferation is enhanced by moderate hypoxia is consistent with previous findings. The growth of cells from human saphenous vein explants in the presence of 10% FCS was enhanced in a 5% O2 environment compared with 20% O2 (55). Also, exposure to 3% O2 enhanced proliferation of SMC derived from bovine pulmonary artery that were costimulated with either phorbol ester (18, 17) or serum (19). However, bovine SMC did not proliferate when exposed to 3% O2 alone, without serum or phorbol ester. In contrast, 5% O2 induced proliferation of human VSMC under low serum conditions, which maintained cell viability but did not induce proliferation. Thus hypoxia alone is an effective mitogenic stimulus for human VSMC.

Recent studies have documented that arteries are composed of phenotypically diverse subpopulations of SMC and cells with nonmuscle characteristics (20). Four distinct populations of SMC have been identified within the media of bovine pulmonary artery and aorta, based on distinct morphological appearance, differential expression of muscle-specific proteins, and differential localization between the inner, middle, and outer media (21). Exposure to hypoxia in vivo induces proliferation of a meta-vinculin-negative SMC population in bovine neonatal pulmonary arteries (62). Four distinct cell types can also be isolated from bovine pulmonary artery and aorta in vitro, and these cells retain their characteristics over time in culture. Two of the cell types were SMC-like and proliferated slowly in serum, and moderate hypoxia further inhibited their proliferative rate, whereas the other two types were non-muscle-like and proliferated rapidly in serum, and moderate hypoxia further enhanced their proliferative rate (19). SMC derived from human blood vessels are likewise phenotypically heterogeneous with respect to morphological appearance, growth rates (22), responsivenesss to growth factors (8), and expression of muscle-specific proteins (25). Thus it seems likely that the mixed cultures of human VSMC used in the present study contain subpopulations of cells that respond differentially to hypoxia.

Levels of cell-associated IL-1alpha were not increased in human VSMC incubated in 5% O2. Studies with exogenous IL-1ra further ruled out the possibility that the proliferative response to 5% O2 involves a membrane-associated form of IL-1alpha that may not be detected by EIA analysis of cell lysates. Exogenous IL-1ra, by binding to the type I IL-1 receptor, can inhibit the action of either IL-1, which is released by cells, or membrane-associated IL-1 (32); however, higher concentrations of IL-1ra are required to inhibit the membrane-associated form. In the present study, high concentrations of exogenous IL-1ra did not affect proliferation of human VSMC incubated in either low, 5%, or 20% O2, ruling out a role for released IL-1 (alpha  or beta ) or membrane-associated IL-1alpha in human VSMC proliferation in low or normal O2 tension. Although the studies with exogenous IL-1ra do not rule out a possible intracellular action of IL-1alpha , this possibility seems unlikely, since IL-1alpha was not detectable in the lysates of human VSMC incubated in 5% O2. These results demonstrate two direct and distinct effects of hypoxia on VSMC. Exposure to intermediate O2 tension induces VSMC proliferation, independent of IL-1, whereas exposure to low O2 tension induces production of IL-1alpha .

We have previously reported that HSVSMC produce an icIL-1ra (6) that lacks the signal peptide sequence found in the secreted form of IL-1ra (sIL-1ra) and remains cell associated. Production of icIL-1ra is augmented in human VSMC that have been stimulated with exogenous IL-1, phorbol ester, platelet-derived growth factor, or transforming growth factor-beta (6). In the present study, exposure to low O2 induced icIL-1ra gene expression in HPASMC; however, the effect was modest, and the amount of IL-1ra produced (1 ng/106 cells) was low compared with that produced by HSVSMC that have been stimulated with IL-1 or phorbol ester (6-14 ng/106 cells; see Ref. 6). icIL-1ra, in contrast to sIL-1ra, has been proposed to act as a unique intracellular inhibitor that attenuates IL-1 signaling at a point downstream from the IL-1 receptor in cancer cell lines (59). It is not known whether icIL-1ra modulates IL-1 signaling in VSMC or whether the modest amounts of icIL-1ra that are produced during hypoxia would be effective.

Chronic alveolar hypoxia is a common cause of pulmonary hypertension (27), and hypertrophy and hyperplasia of VSMC within the media of distal pulmonary arteries is a common feature of this disease (50, 44). Systemic hypoxia may also aggravate the development of atherosclerotic lesions. Cigarette smoking (52), carbon monoxide exposure (1), and chronic sleep apnea (16) are associated with systemic hypoxia and increased incidence of atherosclerosis. Experimentally induced arterial hypoxia enhances the development of atherosclerotic lesions in cholesterol-fed rabbits (34), whereas arterial hyperoxia inhibits lesion development (33). Because both EC and VSMC become hypoxic in the setting of alveolar or systemic hypoxia, both cell types may release VSMC mitogens that contribute to the pathophysiological response, including IL-1, basic fibroblast growth factor, and promitogenic prostanoids (45, 54).

Local hypoxia has also been proposed to be a contributing factor to the development of atherosclerotic lesions, since intimal thickening itself impairs diffusion of O2 from the vessel lumen. Also, O2 consumption is increased in diet-induced atherosclerotic lesions of rabbits (61), most likely due to increased consumption in foam cells in which cholesteryl esters are continually hydrolyzed and reesterified (9, 11). It is possible that local hypoxia can also initiate the development of atherosclerotic lesions, since occlusion or removal of the vasa vasorum in experimental animals results in intimal hyperplasia (47, 43, 2, 3). It has been proposed that human atherosclerosis may be enhanced when a thrombus forms within the vasa vasorum, leading to hypoperfusion and hypoxia of the adventitia and ultimately to the release of cytokines that induce SMC proliferation (43). Because high O2 consumption by foam cells, intimal thickening, or thrombosis within the vasa vasorum would lead to local hypoxia within the vessel wall, and EC would remain well-oxygenated, direct effects of hypoxia on VSMC may predominate in this setting.

In summary, the present studies provide evidence that hypoxia directly stimulates proliferation of human VSMC. Hypoxia-induced VSMC proliferation may contribute to the pathophysiological effects of chronic hypoxia on the vasculature and thus may play a role in the development of pulmonary hypertension or atherosclerosis. Exposure to moderate hypoxia induced human VSMC proliferation without inducing IL-1alpha production, whereas exposure to severe hypoxia induced IL-1alpha production. Hypoxia-induced IL-1alpha production by human VSMC may contribute to the pathophysiological effects of hypoxia on blood vessels.


    ACKNOWLEDGEMENTS

We acknowledge Dr. Paul Hassoun (Pulmonary Division, Department of Medicine, New England Medical Center Hospitals) for critical review of the paper. Human recombinant IL-1ra and reagents for IL-1ra RIAs were kindly provided by Dr. Charles A. Dinarello (Division of Infectious Diseases, University of Colorado Health Sciences Center, Denver, CO).


    FOOTNOTES

This work was supported by Grant HL-47569 from the National Institutes of Health.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. Beasley, New England Medical Center, Box 172, 750 Washington St., Boston, MA 02111 (E-mail: debbie.beasley{at}es.nemc.org).

Received 28 July 1998; accepted in final form 19 May 1999.


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DISCUSSION
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Am J Physiol Heart Circ Physiol 277(4):H1326-H1337
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