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【摘要】
Objectives- Drug eluting stents (DES) reduce the incidence of restenosis after coronary angioplasty. Enthusiasm has been tempered by a possible increased risk of in-stent thrombosis. We examined the effects of paclitaxel and rapamycin on the endothelial transcriptome to identify alterations in gene expression associated with thrombosis.
Methods and Results- Gene expression profiling was performed on human coronary artery endothelial cells treated with rapamycin or paclitaxel. Plasminogen activator inhibitor-1 (PAI-1) was the most consistently induced transcript in rapamycin-treated human coronary artery endothelial cells. RT-PCR and ELISA were performed to confirm positive findings. Transgenic mice engineered to express enhanced green fluorescent protein under control of the human PAI-1 promoter were also treated. Rapamycin and paclitaxel treated endothelial cells produced dose-dependent increases in PAI-1. There was a variable effect on endothelial tissue-type plasminogen activator (t-PA) expression. Enhanced expression of PAI-1 and enhanced green fluorescent protein were detected in coronary arteries, the aorta, and kidney of the mice.
Conclusion- Antiproliferative agents stimulate the expression of prothrombotic genes. PAI-1 expression may also play a role in the prevention of restenosis through an antimigratory mechanism. The effects of antiproliferatives on vascular gene expression deserve further scrutiny in view of the increasing utilization of drug-eluting stents.
To examine the prothrombotic effects of antiproliferative agents on endothelial gene expression, coronary endothelial cells were treated with paclitaxel or rapamycin. Transcriptional profiling was performed and confirmed by RT-PCR, ELISA, and a murine model. Rapamycin and paclitaxel induced increases in endothelial PAI-1 in the coronary arteries and aortas of mice.
【关键词】 antiproliferative plasminogen activator inhibitor rapamycin paclitaxel gene array endothelial cells gene expression
Introduction
Restenosis is widely recognized as a major factor limiting the long-term clinical efficacy of percutaneous coronary intervention. 1 The 30% to 50% incidence of restenosis associated with balloon angioplasty alone has been reduced in half by the use of bare metal stents. 2 Numerous mechanical and pharmacological strategies have failed to further reduce restenosis until recently. 3,4 The development of drug eluting stents (DES) that release small amounts of antiproliferative agents locally provides an effective strategy for reducing the risk of restenosis after angioplasty to levels below 10%. 5,6 These impressive effects on restenosis were tempered initially by concerns about possible increases in the rate of in stent thrombosis with DES. 7,8 Follow-up data from SIRIUS, and other recent trials appeared to alleviate these concerns. 9,10 Since the presentation of these findings, however, the concerns regarding these agents enhancing in stent thrombosis resurfaced. An observational study demonstrated a 30% incidence of in stent thrombosis associated with DES and clopidogrel noncompliance. 11 In addition, there was a great amount of concern regarding late stent thrombosis generated by papers presented at the European Society of Cardiology Meeting in Barcelona this past September. 12 See page 261
There are 2 agents used in drug eluting stents in the United States. Rapamycin binds to the mammalian target of rapamycin (mTOR), arresting division of vascular smooth muscle cells. 13 Paclitaxel stabilizes microtubules in the cytosol resulting in disruption of cell division and migration. 14 There is scant evidence in the literature of a prothrombotic effect of systemic administration of paclitaxel or rapamycin beyond case reports of thrombotic events associated with docetaxel and rapamycin. 15,16 Conversely, rapamycin has been prescribed to transplant recipients who develop calcineurin inhibitor-associated thrombotic microangiopathy because it is believed to be less prothrombotic than cyclosporin. 17 Mechanistically, it is plausible to consider the possibility that rapamycin does have prothrombotic potential, and indeed this agent has recently been demonstrated to increase tissue factor expression in cultured endothelial cells. 18 Paclitaxel has also been demonstrated to enhance thrombin-induced tissue factor expression in endothelial cells through activation of a c-Jun kinase in endothelial cells. 19
Although the antiproliferative and antimitotic effects of DES are believed to be responsible for preventing restenosis, the effects of these agents on global endothelial gene expression are not known. Indeed, if increased tissue factor expression is a potential factor contributing to in stent thrombosis, it is certainly plausible that other prothrombotic alterations in endothelial gene expression may occur in response to exposure to these agents. In this study, we use microarray technology to more broadly investigate potential prothrombotic effects of rapamycin and paclitaxel on cultured human coronary artery endothelial cells.
Methods
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were isolated by using the method of Jaffe et al and cultured in medium199 (M199) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 µg/mL streptomycin, 250 ng/mL amphotericin B (PSA), 30 µg/mL endothelial cell mitogen, 20 U/mL heparin, and 2 mmol/L L-glutamine. 20 Human coronary artery endothelial cells (HCAECs; Cambrex, Walkersville, Md) were grown in EGM-MV medium (Cambrex). Human aortic smooth muscle cells (HASMs; Cambrex) were grown in M199, 15% FBS+PSA. All cells were incubated under 5% CO 2 at 37°C. Cells were passaged by trypsin/EDTA at a split ratio of 1:3 and cultured on gelatin-coated dishes.
Treatment of Endothelial Cells With Antiproliferative Agents
For gene array experiments, HCAECs were grown on 150-mm dishes. Cells were treated with paclitaxel (100 nmol/L; Sigma Chemical Company) or 0.1% ethanol vehicle for 1 week or rapamycin (2.2 nmol/L) (Calbiochem, San Diego, CA) or 0.1% DMSO vehicle for 1 week.
RNA Extraction, Purification, and cDNA Microarray
Cells were lysed and total RNA was isolated using an RNeasy Midi-kit (Qiagen). RNA isolations were performed independently for 6 replicates in each of the 2 treatment groups. The pooling of RNA samples was avoided to allow for the ability to detect any biological variability between individual samples within a treatment group. Each microarray chip experiment profiled RNA from a single culture dish of rapamycin or paclitaxel-treated endothelial cells compared with the RNA from a single dish of vehicle-treated cells. The hybridization process is described in a supplementary Methods section (available online at http://atvb.ahajournals.org).
Microarrays containing 11 520 cloned genes from the human sequence verified clone set from Research Genetics were generated by the Vanderbilt Microarray Shared Resource. Gene lists and protocols are available at http://array.mc.vanderbilt.edu. All cDNA clones were amplified and verified by gel electrophoresis.
Real Time Quantitative RT-PCR
HCAECs in 100-mm plates (n=3, for each cell type and dose), were treated with 0.1% ethanol or various concentrations of paclitaxel dissolved in 0.1% ethanol, or 0.1% DMSO or various concentrations of dissolved in 0.1% DMSO for 1 week. Cells were lysed and total RNA was isolated using an RNeasy Mini-kit (Qiagen). RT-PCR was performed to determine the amount of PAI-1 and t-PA RNA present relative to ß-actin. The details of the methodology are described in a supplementary methods section. Relative quantitative analysis was performed with the 2 - CT method. 21
PAI-1 and t-PA Antigen Assay
To confirm that the transcriptional effects seen by consecutive experiments of microarray and RT-PCR affected cellular protein production and secretion, cells were treated with either rapamycin or paclitaxel in 12-well plates for 72 hours at 37°C. Cells were washed with phosphate buffered saline 3 x before the 48-hour medium change. Because paclitaxel caused a decrease in cell confluence in endothelial and vascular smooth muscle cells, cells were trypsinized, resuspended in phosphate buffered saline, and counted in a cytometer after the conditioned media were sampled. These data were normalized to the number of cells per well. Human PAI-1 and t-PA antigen were measured by colorimetric enzyme linked immunosorbent assay ELISA (Biopool).
Murine Infusion Experiments and Tissue Preparation and Immunohistochemical Staining
Age, sex, and litter matched transgenic mice containing 3KB of the human PAI-1 (3KB-hPAI-1) promoter linked to enhanced green fluorescence protein (eGFP) (n=6 for each treatment group) were loaded with rapamycin (0.50 mg/kg), paclitaxel (1.1 mg/kg), or vehicle (5% dextrose, 0.5% ethanol, 0.5% Cremophor EL) by intraperitoneal injection. 22 The mice were then anesthetized with phenobarbital and had osmotic minipumps placed to allow for the delivery of rapamycin (0.25 mg/kg/d), paclitaxel (0.55 mg/kg/d), or vehicle (5% dextrose, 5% ethanol, 5% Cremophor EL) for 2 weeks. The concentration of rapamycin in the mouse model has previously been shown to inhibit restenosis in an in vivo porcine balloon injury model. 5 The concentration of paclitaxel used was proportional to the systemic rapamycin dose adjusted by the molar ratio of rapamycin to paclitaxel used on the Cypher and Taxus drug eluting stents, respectively. The animals were euthanized at 2 weeks, and the organs were harvested for immunohistochemical analysis to determine the level of expression of murine PAI-1 (mPAI-1) and eGFP. Detailed methodology is included in a supplemental methods section.
Statistical Analysis
We identified genes with significantly altered expression on microarray using the significance analysis of microarrays (SAM) algorithm. 23 A 10-nearest neighbor imputation engine was used to analyze one class response data. The false discovery rate for all arrays was less than 0.05, the false significant number was less than 0.5, and the delta was less than 1.9 for all experimental sets. Array results are reported in tables as the significance score (d) and the associated q value. The significance score is a value that is based on the change in gene expression between the treated and untreated samples relative to the standard deviation of repeated measurements for that gene. The q value is the lowest false discovery rate at which a gene is called significant, similar to a probability value, but adapted to the analysis of a large number of genes.
PAI-1 is reported in the text as the log 2 of the ratio of medians±SD. The ratio of medians is a ratio of the median measurement of Cy5 and Cy3 fluorescence for a spot on an array.
RT-PCR results are presented as relative induction. PAI-1 antigen secretion rates are presented as picograms/2 x 10 5 cells per hour (pg/2 x 10 5 cells per hour). Dose response curves for paclitaxel and rapamycin treatment for RT-PCR, and antigen measurements were analyzed using one-way ANOVA with statistical significance at the five percent level. All results are shown as the mean±SEM.
Results
Effects of Antiproliferative Agents on the Transcriptome of HCAECs
In rapamycin-treated HCAECs, there were a total of 1354 transcripts (12.3%) identified as showing significant alteration versus control, with 889 (8.0%) induced and 465 (4.2%) reduced using a false significant number of 0.40311 with a delta of 1.23219 (supplemental Table I). The group of induced transcripts included PAI-1, metallothioneins 1 B,F,G,H, and X, t-PA, hexokinase, endothelial cell specific molecule, interleukin (IL)-1 receptor-like protein 1, regulator of G-Protein signaling 4, keratin 19, AXL receptor tyrosine kinase, and hyaluronoglucosaminidase 2 (supplemental Table Ia). Prominent among gene transcripts downregulated by rapamycin were chemokine ligand CC15, osteoblast specific factor 2, CD34 antigen, multimerin, fatty acid binding protein 4, pyruvate dehydrogenase kinase isoenzyme 4, matrilin 2, phosphodiesterase 2, von Willebrand factor (vWF), and bone morphogenic protein 4 (supplemental Table Ib).
Initial experiments using 10 µmol/L doses of paclitaxel resulted in essentially no significant alterations in gene expression (data not shown), so experiments were repeated using the higher dose of 100 µmol/L. In paclitaxel treated HCAECs, a total of 850 transcripts (7.7%) were induced and 235 (2.1%) were reduced compared with controls using a false significance number of 0.42176 and a delta of 1.82135 (supplemental Table II). The genes showing the greatest relative induction were tubulin -1 and 2, and tubulin ß-5, chemokine ligands CC 2 and 7, phospholipid transfer protein, MHC Class-1C, cytoplasmic FMR-1 interacting protein 2, signal transducer and activator of transcription 1, serum-inducible kinase, PAI-1 and t-PA (supplemental Table IIa). Downregulated genes included phosphodiesterase-2, high mobility group nucleosomal binding domain 3, pirin, chemokine ligand CC 3, pyruvate dehydrogenase kinase isoenzyme 4, tissue factor pathway inhibitor, ATP-binding cassette subfamily A, member 6, and nuclear transcription factor Y-ß. (supplemental Table IIb).
PAI-1 and t-PA were significantly positively altered in both gene array surveys performed on HCAECs. The mean log 2 ratios were 3.10±0.22 and 2.54±0.07 for PAI-1 and t-PA in rapamycin-treated HCAECs and 2.08±0.46 and 1.80±0.46 in paclitaxel-treated HCAECs.
Effects Rapamycin on PAI-1 and t-PA Production in Endothelial and Vascular Smooth Muscle Cells
Coronary artery and umbilical vein endothelial cells treated with rapamycin demonstrated dose dependent increases in PAI-1 and dose dependent decreases in t-PA message expressions that were statistically significant. In aortic smooth muscle cells, there was a trend toward a decrease in PAI-1 message expression and a significant decrease in t-PA message expression ( Figure 1A and 1 B).
Figure 1. A, Effects of rapamycin on relative PAI-1 mRNA expression (Fold Expression). HCAECs (- -), HUVECs (- -), HASMs (- -); ANOVA for trend, * P =0.008, P <0.001, NS, P =Not significant. B, Effects of rapamycin on relative t-PA mRNA expression (Fold Expression). HCAECs (- -), HUVECs (- -), HASMs (- -), ANOVA for trend, * P =0.002, P <0.001. C, Effects of rapamycin on PAI-1 protein secretion (pg/2 x 10 5 cells per hour). HCAECs (- -), HUVECs (- -), HASMs (- -), ANOVA for trend, * P =0.02, P =0.008, P =0.05. D, Effects of rapamycin on t-PA protein secretion (pg/2 x 10 5 cells per hour). HCAECs (- -), HUVECs (- -), HASMs (- -), ANOVA for trend, * P =0.03, P =0.008, P =0.002.
PAI-1 antigen secretion was increased in a dose dependent manner in coronary artery endothelial cells but decreased in aortic smooth muscle cells ( Figure 1 C). In all three cell types, t-PA secretion was reduced in a dose-dependent manner ( Figure 1 D). The protein data for HCAECs treated with 22 nmol/L rapamycin 75% cell dropout). RNA experiments were not curtailed by this cytotoxicity.
Effects Paclitaxel on PAI-1 and t-PA Production in Endothelial and Vascular Smooth Muscle Cells
PAI-1 and t-PA message expression was increased in all 3 cell types in a dose-dependent manner ( Figure 2A and 2 B). PAI-1 antigen secretion was increased in both endothelial cell types and slightly decreased in aortic smooth muscle cells ( Figure 2 C). Secretion of t-PA decreased in coronary artery endothelial cells but increased in umbilical vein endothelial cells. There was essentially no change in t-PA secretion in aortic smooth muscle cells in response to paclitaxel treatment ( Figure 2 D).
Figure 2. A, Effects of paclitaxel on relative PAI-1 mRNA expression (Fold Expression). HCAECs (- -), HUVECs (- -), HASMs (- -); ANOVA for trend, * P <0.001. B, Effects of paclitaxel on relative t-PA mRNA expression (Fold Expression). HCAECs (- -), HUVECs (- -), HASMs (- -), ANOVA for trend, * P <0.001, P =0.0031. C, Effects of paclitaxel on PAI-1 protein secretion (pg/2 x 10 5 cells per hour). HCAECs (- -), HUVECs (- -), HASMs (- -), ANOVA for trend, * P <0.001, P =0.003. D, Effects of paclitaxel on t-PA protein secretion (pg/2 x 10 5 cells per hour). HCAECs (- -), HUVECs (- -), HASMs (- -), ANOVA for trend, * P =0.03, P <0.001, NS, P =Not significant.
The Effects of Rapamycin and Paclitaxel on Murine PAI-1 and 3KB-hPAI-1 Promoter-Driven eGFP Expression
Continuous infusion of rapamycin and paclitaxel resulted in an increased expression of murine PAI-1 in coronary arteries compared with vehicle ( Figure 3A through 3 C). This pattern was also seen in the aorta ( Figure 4A through 4 C), and in the kidney ( Figure 5A through 5 C). There was also increased expression of eGFP in the coronary arteries ( Figure 3D through 3 F), aorta ( Figure 4D through 4 F), and kidney ( Figure 5D through 5 F), demonstrating activation of the human PAI-1 promoter in both rapamycin and paclitaxel-treated mice, compared with vehicle-treated mice.
Figure 3. These are representative photomicrographs of murine cardiac tissue taken at 40 x. Mice were treated with vehicle (A, D), rapamycin (B, E), and paclitaxel (C, F). Immunostaining was performed for murine PAI-1 (A-C) and eGFP (D-F).
Figure 4. These are representative photomicrographs of murine aortic tissue taken at 20 x. Mice were treated with vehicle (A, D), rapamycin (B, E), and paclitaxel (C, F). Immunostaining was performed for murine PAI-1 (A-C) and eGFP (D-F).
Figure 5. These are representative photomicrographs of murine renal tissue taken at 20 x. Mice were treated with vehicle (A, D), rapamycin (B, E), and paclitaxel (C, F). Immunostaining was performed for murine PAI-1 (A-C) and eGFP (D-F).
Discussion
There is a relative paucity of data on the impact of antiproliferative agents on vascular gene expression. In this study, we used gene array technology to perform an unbiased survey of the effects of rapamycin and paclitaxel on endothelial gene expression with a focus on identifying transcripts that might be associated with thrombosis. Despite the potent antiproliferative effects of rapamycin and paclitaxel, we consistently observed changes in less than 15% of the 11 000 genes placed on a microarray. Interestingly, among the 200 most significantly altered genes, the only genes altered in HCAECs by both antiproliferative agents included PAI-1, beta 5-tubulin, endothelial cell-specific molecule-1, metallothioneins 1H and 1L, and metastasis associated protein-1.
The most striking and consistent effect of the antiproliferative agents was the effect on PAI-1 expression and secretion. Endothelial PAI-1 expression and secretion increased in the presence of rapamycin, as did endothelial and aortic smooth muscle expression and endothelial secretion in the presence of paclitaxel. These findings are further emphasized and validated in vivo, as we observed a similar pattern of increased expression of murine PAI-1 and human PAI-1 promoter activity in the coronary arteries, aorta, and kidney of transgenic mice treated with both paclitaxel and rapamycin. Rapamycin also caused decreased PAI-1 expression and secretion in human aortic smooth muscle cells and in t-PA expression and secretion in all 3 cell types studied.
Paclitaxel treatment resulted in increased endothelial and aortic smooth muscle PAI-1 expression; however, paclitaxel had no apparent effects on smooth muscle cell secretion of PAI-1 and t-PA and HCAEC secretion of t-PA. This discrepancy between message expression and protein secretion in these experiments may reflect the disruption of microtubules with impaired secretion of PAI-1 and t-PA via regulated secretory pathways. 24-26
The present findings suggest that if there is an increased tendency toward in stent thrombosis in patients that receive DES, some of the risk may be mediated via local increases in PAI-1 without commensurate increases in local t-PA release. There are, however, several caveats to this suggestion and limitations to this study. First, the effects of the antiproliferative agents seen in this study may not accurately reflect the local concentrations of antiproliferatives as they elute from the coated stent in vivo. Similarly, the murine experiments described here used systemic doses of drug comparable to doses administered in a previously reported porcine model rather than extrapolating the theoretical tissue concentrations of the drugs. 5
The work described here was primarily an in vitro investigation of endothelial monolayers, and may not represent the complex biology of these antiproliferative agents on coronary endothelial expression in an atherosclerotic lesion after stenting. The murine model, though more complex, was free of atherosclerotic disease. Furthermore, the murine model did not involve balloon-mediated vessel injury. Finally, because we were screening for drug effects in vascular cells in vitro, we typically examined effects on transcriptional profiles using 6 replicates per group. This experimental design is admittedly exploratory and does not provide the statistical power used to predict false and expected discovery rates typically seen in studies analyzing 50 or 100 chip experiments. For this reason, we designed and performed additional experiments that demonstrated the effects of the agents on PAI-1 expression and secretion in cultured endothelial cells and in vivo. However, because we compared individual samples on gene array rather than replicates of pooled RNA, significant biological variability between replicates or any other systematic bias resulting from the independent samples used for the array experiments would have been apparent.
Although this study does not describe the mechanism by which rapamycin and paclitaxel induce gene expression, there are potential mechanisms described that may explain the effects of these antiproliferative agents on PAI-1 expression. Rapamycin is known to inhibit E2F, which is reported to act as a repressor of PAI-1 gene expression, and it is potentially through this pathway that PAI-1 expression is upregulated. 27,28 A mechanism through which paclitaxel induces PAI-1 expression is less apparent. Paclitaxel has been shown to alter gene expression through NF- B, AP-1 response elements, and through stabilization of mRNA, all of which may act alone or in combination to increase PAI-1 mRNA levels. 29,30
DES are designed to deliver antiproliferative agents into the local environment for at least several weeks, with a gradual decay in the concentration as the site of injury heals. While systemic levels of the agents are likely quite low, the local tissue levels are undoubtedly closer to those used in the experiments described here. In the clinical setting, DES exert their effects in atherosclerotic arteries after injury in the face of a number of biochemical and mechanical stimuli that undoubtedly contribute to the local proliferative response and the tendency toward in stent thrombosis. Furthermore, aggressive antiplatelet therapy has been demonstrated to reduce any tendency toward thrombosis to that seen with bare metal stenting. 10,31
The clinical impact of alterations in PAI-1 production is uncertain. The increased local production of PAI-1 in response to paclitaxel may partly explain the apparent increased incidence of in stent thrombosis reported in the SCORE Trial. 32 It is uncertain whether, and for how long, increased expression of PAI-1 raises the risk of thrombosis. The upregulation of PAI-1 in HCAECs seen here is particularly worrisome, especially in conjunction with downregulation of t-PA secretion in coronary artery endothelial cells, as t-PA plays as a principal profibrinolytic role in the coronary vasculature. 33 If these effects occur in vivo, the induction of PAI-1 by rapamycin and paclitaxel in the coronary endothelium without adequate t-PA secretion to compensate for it may have important and deleterious consequences on the efficacy of the fibrinolytic system in the coronary vasculature. In practice and in clinical trials, the use of antiplatelet therapies such as clopidogrel, ticlopidine, and aspirin is recommended throughout the period of drug elution and beyond the time in which stents are presumably covered with endothelial cells. 34 Certainly, the recommendation for combined antiplatelet therapy exceeds the recommendations that are conventionally applied for bare metal stents. Aggressive antiplatelet therapy used in recent trials has been reported to reduce the incidence of in stent thrombosis to levels seen with bare metal stents, and continuation of this clinical approach seems prudent and prescient in view of the current findings. 35
From a different perspective, the results presented here suggest another mechanism, beyond the antiproliferative effects, by which DES could limit the development of restenosis. PAI-1 has been shown to retard smooth muscle cell migration, to impair wound healing, and to reduce neointimal proliferation in response to vascular injury in mice. 36 Therefore, an increase in endothelial and vascular smooth muscle PAI-1 production in response to local antiproliferative agents eluting from coated stents may contribute to the prevention of restenosis above and beyond the direct antiproliferative effects of agents such as rapamycin and paclitaxel. Although impaired wound healing may be desired in response to balloon injury of a diseased coronary artery, deleterious wound healing complications have been observed in association with perioperative administration of rapamycin in lung, kidney, and liver transplant recipients. 37-39 Alterations in PAI-1 and other gene products merit further investigation as drug-eluting stents are applied more broadly as the antiproliferative and prothrombotic effects may be magnified or diminished in other vascular beds or in patients that constitutively overproduce PAI-1, such as Type 2 diabetics or patients with the metabolic syndrome. 40
Acknowledgments
Sources of Funding
J.A.S.M. is a research scholar supported by the National Institutes of Health (5T32-HL07411-23) and The Stanley J. Sarnoff Endowment for Cardiovascular Research (Great Falls, Va). This work was also supported by the National Institutes of Health (RO1-HL51387, RO1-HL65192, P50-HL081009, and the Vanderbilt Murine Metabolic Physiology Core supported by NIDDK-59637; D.E.V.). All microarray experiments were performed in the Vanderbilt Microarray Shared Resource. The Vanderbilt Microarray Shared Resource is supported by the Vanderbilt Ingram Cancer Center (P30-CA68485), the Vanderbilt Diabetes Research and Training Center (P60-DK20593), the Vanderbilt Digestive Disease Center (P30-DK58404), and the Genomics of Inflammation Program Project Grant (P01-HL6744-01).
Disclosures
None.
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作者单位：James A.S. Muldowney, III; John R. Stringham; Shawn E. Levy; Linda A. Gleaves; Mesut Eren; Robert N. Piana; Douglas E. VaughanFrom the Division of Cardiovascular Medicine, Department of Medicine (J.A.S.M., J.R.S., L.A.G., M.E., R.N.P., D.E.V.), the Department of Molecular Physiology and Biophysics (