MicroRNA Expression Signature and Antisense-Mediated Depletion Reveal an Essential Role of MicroRNA in Vascular Neointimal Lesion Formation
MicroRNAs (miRNAs) are a recently discovered class of endogenous, small, noncoding RNAs that regulate about 30% of the encoding genes of the human genome. However, the role of miRNAs in vascular disease is currently completely unknown. Using microarray analysis, we demonstrated for the first time that miRNAs are aberrantly expressed in the vascular walls after balloon injury. The aberrantly expressed miRNAs were further confirmed by Northern blot and quantitative real-time polymerase chain reaction. Modulating an aberrantly overexpressed miRNA, miR-21, via antisense-mediated depletion (knock-down) had a significant negative effect on neointimal lesion formation. In vitro, the expression level of miR-21 in dedifferentiated vascular smooth muscle cells was significantly higher than that in fresh isolated differentiated cells. Depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose-dependent manner. MiR-21–mediated cellular effects were further confirmed in vivo in balloon-injured rat carotid arteries. Western blot analysis demonstrated that PTEN and Bcl-2 were involved in miR-21–mediated cellular effects. The results suggest that miRNAs are novel regulatory RNAs for neointimal lesion formation. MiRNAs may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke.
MicroRNAs (miRNAs) are endogenous, noncoding, single-stranded RNAs of ≈22 nucleotides and constitute a novel class of gene regulators.1–3 Although the first miRNA, lin-4, was discovered in 1993,4,5 their presence in vertebrates was confirmed only in 2001.6 MiRNAs are initially transcribed by RNA polymerase II (Pol II) in the nucleus to form large pri-miRNA transcripts.7 The pri-miRNAs are processed by the RNase III enzymes, Drosha and Dicer, to generate 18- to 24-nucleotide mature miRNAs. The mature miRNAs negatively regulate gene expression in 1 of 2 ways that depend on the degree of complementarity between the miRNA and its target. MiRNAs that bind to 3′UTR of mRNA with imperfect complementarity block protein translation. In contrast, miRNAs that bind to mRNA with perfect complementarity induce targeted mRNA cleavage. Currently, more than 400 miRNAs have been cloned and sequenced in human, and the estimated number of miRNA genes is as high as 1000 in the human genome.8,9 As a group, miRNAs are estimated to regulate 30% genes of the human genome.10 Analogous to the first RNA revolution in the 1980s with Cech discovering the enzymatic activity of RNA,11 this recent discovery of RNAi and miRNA may represent the second RNA revolution.12
Small interfering RNAs (siRNAs) are another class of small noncoding RNAs that have similar mechanism for gene expression regulation. However, they are different from each other.5,13 The chief difference lies in their origins.4,13 SiRNAs are produced from long, double-stranded (bimolecular) RNAs or long hairpins, often of exogenous origin, and usually target sequences at the same locus or elsewhere in the genome for destruction (gene silencing).14,15 This phenomenon is termed RNAi.16 In contrast, miRNAs are endogenous. They are encoded within the genome and come from endogenous short hairpin precursors and usually target sequences at other loci. Therefore, miRNAs are important endogenous regulators for gene expression.
The biological roles of only a small fraction of identified miRNAs have been elucidated to date. In fact, we are just beginning to understand how this novel class of gene regulators is involved in biological functions. Although only a small number of the hundreds of identified miRNAs have been characterized, a growing body of exciting evidence suggests that miRNAs are important regulators for cell growth, differentiation, and apoptosis.13,17,18 Therefore, miRNAs may be important for normal development and physiology. Consequently, dysregulation of miRNA function may lead to human diseases.19 In this respect, the most exciting research area is the role of miRNAs in cancer, given that cell dedifferentiation, growth, and apoptosis are important cellular events in the development of cancer. Indeed, both basic and clinical studies have demonstrated that miRNAs are aberrantly expressed in diverse cancers.20–23 MiRNAs are currently thought to function as both tumor suppressors and oncogenes.24
Cardiovascular disease has long been the leading cause of death in developed countries, and it is rapidly becoming the number 1 killer in developing countries.25 Neointimal formation is a common pathological lesion in diverse cardiovascular diseases such as atherosclerosis, coronary heart diseases, postangioplasty restenosis, and transplantation arteriopathy. Although miRNAs are expressed in the cardiovascular system,26 the roles of these miRNAs in vascular diseases are completely unknown. As neointimal lesion formation in proliferative vascular disease shares similar cellular events and molecular mechanisms with cancers,27 we therefore hypothesized that miRNAs might play important roles in neointimal lesion formation.
Materials and Methods
An expanded Materials and Methods section is provided in the online data supplement at http://circres.ahajournals.org.
Rat Carotid Artery Balloon Injury Model
Carotid artery balloon injury was induced in male Sprague-Dawley rats (250 to 300 g) (Harlan Sprague-Dawley, Inc, Indianapolis, Ind) as described.28
Local Oligo Delivery
miRNA inhibitors were delivered into injured vascular tissue using an established protocol.29
Morphometric Analysis for Neointimal Lesion Formation
Morphometric analysis was performed in carotid artery sections as described.28
Vascular smooth muscle cells (VSMCs) were obtained from the aortic media of male Sprague-Dawley rats (5 weeks old) as described.30
Oligo Transfection, miR-21 Knockdown, and miR-21 Overexpression in Cultured VSMCs
Oligo transfection in cultured cells was performed according to an established protocol.31,32
VSMC apoptosis was measured by dUTP nick-end labeling (TUNEL) assay, caspase-3 activation measurement, and immunohistochemistry.33,34
Cell Viability Assay
Cell viability was measured by bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method.35
Gene Microarray Analysis for miRNA Expression
MiRNA expression profiling was determined by miRNA microarray analysis by using the rat miRNA array probes (LC Sciences).
Quantitative Real-Time Polymerase Chain Reaction
MiRNA levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) with the mirVana miRNA detection kit (Ambion, Inc).
Northern Blot Analysis of miRNA
The probe sequences of these miRNAs are shown in supplemental Table I.
Western Blot Analysis
Phosphatase and tensin homology deleted from chromosome 10 (PTEN), Akt, phosphorylated Akt, and B-cell leukemia/lymphoma 2 (Bcl-2) levels were determined by Western blot analysis.
MiRNAs Are Highly Expressed in Rat Carotid Artery
Tissue-specific expression is one important characteristic of miRNA expression.26 Indeed, one miRNA may be highly expressed in one tissue but have no or low expression in other tissues.26 To study the biological functions of miRNA in vascular disease, we determined the miRNA expression profile in rat carotid arteries through miRNA microarray analysis. Overall, 140 miRNAs of the 180 arrayed were found in normal rat carotid arteries; 49 of the 140 were highly expressed in normal arteries (supplemental Table II).
MiRNAs Are Aberrantly Expressed in Rat Carotid Arteries After Angioplasty
To determine the expression changes of miRNAs in vascular wall with neointimal formation, we applied the well-established rat carotid artery balloon injury model.28 Interestingly, compared with normal uninjured arteries, microarray analysis demonstrated that aberrant miRNA expression was a remarkable characteristic in vascular walls after angioplasty. Seven days after balloon injury, 113 of the 140 artery miRNAs were differentially expressed with probability value <0.01; 60 miRNAs were upregulated, and 53 miRNAs were downregulated. At 14 days after injury, 110 of the 140 artery miRNAs were differentially expressed (63 up and 47 down), whereas 102 of the 140 artery miRNAs were differentially expressed (55 up and 47 down) at 28 days after angioplasty. Figure 1A shows the time course changes of miRNAs that were highly expressed in rat carotid artery and more than 1-fold upregulated after angioplasty. Figure 1B shows the time course changes of miRNAs that were highly expressed in rat carotid artery and more than 50% downregulated after angioplasty. All the differentially expressed miRNAs at these 3 time points are listed in supplemental Table III.
Aberrant Expression of miRNAs in Injured Arteries Is Confirmed by qRT-PCR or Northern Blot Analysis
These miRNAs whose expression was significantly dysregulated based on microarray analysis were selected for confirmation by qRT-PCR or Northern blot. In agreement with the results from microarray analysis, we found that miR-21, 146, 214, and 352 were highly upregulated, whereas miR-125a, 125b, 133a, 143, 145, 347, and 365 were significantly downregulated during different time courses. Remarkably, miR-21 had more than a 5-fold increase compared with the control. (Figure 1C and 1D). Therefore, miR-21 was further studied using our in vitro and in vivo models to determine the potential biological function of these aberrantly expressed miRNAs.
Downregulation of Overexpressed miR-21 Decreases Neointima Formation in Rat Carotid Artery After Angioplasty
To determine the potential roles of aberrantly expressed miRNAs in neointimal lesion formation after angioplasty, we applied antisense oligonucleotide-mediated miRNA depletion to knock down an overexpressed miRNA, miR-21. The antisense oligonucleotide for miR-21 was modified at each nucleotide by an O-methyl moiety at the 2′-ribose position. The modified antisense oligonucleotide (2′OMe-miR-21) is also called miRNA inhibitor. 2′OMe-miR-21 is synthesized by Integrated DNA Technologies and has the following sequence and structure: 5′mUmCmAmAmCmAmUmCmAmGmUmCmUmGmAmUmAmAmGmCmUmA-3′.36,37 We used 2 controls for this study. The first control was a vehicle control (PBS), and the second control was the modified antisense oligonucleotide for enhanced green fluorescence protein (EGFP) mRNA (2′OMe-EGFP). EGFP gene is a mutant form of green fluorescence protein (GFP) gene.38 Neither EGFP and GFP genes are expressed in rats and transfection of exogenous EGFP, and GFP has no effect on VSMC growth and vascular neointimal formation.39,40 Thus, 2′OMe-EGFP targeting EGFP mRNA is used as a negative oligonucleotide control.37 2′OMe-EGFP is also synthesized by Integrated DNA Technologies and has the following sequence and structure: 5′-mAmAmGmGmCmAmAmGmCmUmGmAmCmCmCmUmGmAmAmGmU-3′.36
As shown in Figure 2B, the fluorescent-marked 2′OMe-miR-21 and 2′OMe-EGFP were successfully delivered into the vascular wall after injury by using the local oligo delivery system (Figure 2A). Consistent with the fluorescent activity analysis, 2′OMe-miR-21 decreased miR-21 expression significantly in the vascular walls at 3 and 7 days after balloon injury (Figure 2C). In contrast, 2′OMe-EGFP had no effect on miR-21 expression (Figure 2C). 2′OMe-miR-21–induced miRNA inhibition is miR-21 specific, as no inhibitory effect was found on other miRNAs such as miR-24 and miR-146 (supplemental Figures I and II).
To determine the effect of the miRNA inhibitor 2′OMe-miR-21 on neointima formation and vascular remodeling, the injured carotid arteries were isolated 14 days after treatment for morphometric analysis. We found that downregulation of the overexpressed miR-21 inhibited neointima formation in rat carotid artery after angioplasty (Figure 2D, 2E, and 2F). In contrast, 2′OMe-EGFP had no effect on neointima formation. Representative hematoxylin-eosin (H-E) stained photomicrographs of rat carotid arteries from different groups are shown in Figure 2D.
Inhibition of miR-21 Decreases Proliferation of Cultured VSMCs
VSMC proliferation is the key cellular event for neointimal lesion formation. To identify the cellular mechanism involved in miR-21–mediated effect on neointimal lesion formation, we determined the effect of miR-21 inhibitor 2′OMe-miR-21 on cell proliferation in cultured VSMCs in the following experiments.
It is well known that freshly isolated VSMCs mimic differentiated VSMCs in a normal uninjured vascular wall, whereas serum cultured VSMCs mimic dedifferentiated VSMCs in vascular neointimal lesions. We thus determined the miR-21 levels in freshly isolated differentiated VSMCs and dedifferentiated VSMCs cultured in DMEM containing 10% FBS. We found that the expression of miR-21 in dedifferentiated VSMCs was significantly higher than that in freshly isolated differentiated VSMCs (Figure 3A). The in vitro result is consistent with the in vivo results in normal and balloon-injured arteries (Figures 1 and 2⇑).
To further determine the potential roles of miRNAs in VSMC proliferation, we applied antisense oligonucleotide-mediated miRNA depletion to knock down the miR-21 overexpression by using its inhibitor, 2′OMe-miR-21. Oligonucleotide transfection was performed according to an established protocol.36 As shown in Figure 3B, fluorescent-marked 2′OMe-miR-21 and 2′OMe-EGFP were successfully transfected into the cultured VSMCs. Consistent with the transfection, 2′OMe-miR-21 decreased the miR-21 expression levels (Figure 3C) in a dose-dependent manner, with a significant decrease observed at a concentration of 3 nmol/L and the maximum effect at 100 nmol/L. In contrast, the control oligo, 2′OMe-EGFP, had no effect on miR-21 level, even at the highest concentration (100 nmol/L). In addition, 2′OMe-miR-21–induced miRNA inhibition in cultured cells is also miR-21 specific, as no inhibitory effect was found on other miRNAs such as miR-24 and miR-146 (Figure 3C).
In subsequent experiments, we determined the effect of 2′OMe-miR-21 on VSMC proliferation by using 2 different methods: cell counting and BrdU incorporation assay as described.41 Consistent with the levels of miR-21 in Figure 3C, 2′OMe-miR-21 significantly decreased cell numbers and BrdU incorporation at 48 hours after culture with DMEM containing 10% FBS (Figure 3D and 3E). Representative BrdU-stained photomicrographs (Figure 3F, bottom panel) as well as their corresponding total cell photomicrographs (Figure 3F, top panel) are shown in Figure 3F. The result indicated that miR-21 has a proproliferative effect on cultured VSMCs. In contrast, 2′OMe-EGFP (100 nmol/L) had no effect on VSMC proliferation.
Inhibition of miR-21 Increases Apoptosis of Cultured VSMCs
Neointimal growth is the balance between cell apoptosis and cell proliferation. Thus, apoptosis is also an important cellular event in neointimal lesion formation. Recent reports demonstrated that miR-21 had an antiapoptosis effect on glioblastoma cells, but had no antiapoptosis effect on HeLa cells.36,37
To determine the role of miR-21 in VSMC apoptosis, we applied a VSMC apoptosis model in which apoptosis was measured after 48 hours in serum-free culture.34 The VSMCs were divided into the following groups: vehicle control, antisense oligo control 2′OMe-EGFP, and miR-inhibitor 2′OMe-miR-21. Apoptosis was evaluated by TUNEL assay and caspase-3 activity measurement. We found that 2′OMe-miR-21 increased TUNEL-positive cells (Figure 4A), accompanied by increasing caspase-3 activity (Figure 4B). Representative TUNEL-stained photomicrographs (Figure 4C, bottom panel), as well as their corresponding total cell photomicrographs (Figure 4C, top panel), are shown in Figure 4C. In contrast, 2′OMe-EGFP (100 nmol/L) had no effect on apoptosis. The results revealed that miR-21 had an anti-apoptotic effect in cultured VSMCs.
To avoid of nonspecific cellular toxicity of miR-21 inhibition, the following two approaches were applied in addition to control oligo 2′OMe-EGFP application. First, we determined the cellular viability using MTT method after transfection of antisense oligonucleotides but before the onset of apoptosis induction. No nonspecific toxicity was found at our experimental concentration range (Figure 4D). Second, we determined the effect of miR-21 inhibition on cell apoptosis without serum deprivation. As shown in Figure 4E, under 10% serum culture condition, few cells underwent apoptosis in the vehicle and 2′OMe-EGFP-treated groups. 2′OME-miR-21 increased the apoptosis rate in a dose-dependent manner. The maximal effect occurred at 100 nmol/L with an apoptosis rate at about 12%. The results indicate that the apoptosis effect is not a nonspecific toxicity. However, miR-21 knockdown and serum deprivation may have a synergetic effect on apoptosis, although the mechanism is currently not clear (Figure 4A, 4B, and 4C).
Inhibition of miR-21 Modulates Cell Proliferation and Apoptosis In Vivo in Injured Rat Carotid Artery
To further determine the cellular effects of miR-21 in vivo, we used immunohistochemistry to determine the proliferation and apoptosis in injured vascular walls as described in our previous publications.31,33 Injured rat carotid arteries treated with vehicle, 2′OMe-EGFP, or 2′OMe-miR-21 were isolated at 7 days after balloon injury. We found that miR-21 inhibitor 2′OMe-miR-21 decreased cell proliferation (Figure 5) and increased apoptosis (Figure 6) in injured vascular walls. In contrast, the control oligo 2′OMe-EGFP had no effect on either proliferation or apoptosis (Figures 5 and 6⇓).
PTEN and Bcl-2 Are Involved in 2′OME-miR-21–Mediated Cellular Effects on VSMCs
To identify the potential molecular targets of miR-21 that may contribute to miR-21–mediated cellular effects, bioinformatics programs such as microrna.sanger.ac.uk, genes.mit.edu/cgi-bin/targetscn, and microran.org were used. Based on the known genes that are involved in smooth muscle cell growth and apoptosis and these potential miR-21 target genes from bioinformatics databases, we propose that PTEN and Bcl-2 might be potential targets for miR-21. As shown in Figure 7, 2′OMe-miR-21 increased PTEN expression (Figure 7A) but decreased Bcl-2 expression (Figure 7B). To further confirm the effects of miR-21 on PTEN and Bcl-2, miR-21 expression was upregulated via transfection of miR-21 (Proligo-Sigma) using the method as described in a recent report.32 We found that miR-21 expression had a 6-fold increase in miR-21 (10 nmol/L) transfected VSMCs compared with those in vehicle and control oligo-treated VSMCs as determined by qRT-PCR. Contrary to miR-21 inhibition, miR-21 overexpression decreased PTEN expressed (Figure 7C) but increased Bcl-2 expression (Figure 7D).
Akt is a downstream signal molecule of PTEN. To further confirm the involvement of PTEN in miR-21–mediated effects, Akt activity was determined. Consistent with the expression changes of PTEN (Figure 7A and 7C), miR-21 inhibition decreased but miR-21 overexpression increased Akt activity (Figure 7A and 7C).
Tissue-specific expression is one important characteristic of miRNA expression.26 With respect to the cardiovascular system, miRNA expression profile in the heart has recently been described.26 In the current study, miRNA expression signature in vessels was uncovered for the first time. Indeed, miRNA expression profile in artery is different from that in heart. For example, the most abundant miRNAs in heart are miR-1, let-7, miR-133, miR-126–3p, miR-30c, and miR-26a26 (our unpublished data). However, in artery the most abundant miRNAs are miR-145, let-7, miR-125b, miR-125a, miR-23, and miR-143. MiR-1 is not an abundant miRNA in artery (data not shown). The different expression profiles in different tissues indicate that the physiological functions of miRNAs in different tissues could be different. Identifying these tissue-specific miRNAs and their physiological functions could be important for future studies.
As a novel class of gene regulators, miRNAs play important roles not only in normal development and physiological conditions, but also in disease status. In this respect, both basic and clinical studies have demonstrated that miRNAs are aberrantly expressed in diverse cancers.19–23 As proliferative vascular diseases share similar cellular events and molecular mechanisms with cancer,27 we hypothesized that miRNAs might also play important roles in these vascular diseases. It is well known that neointimal lesion formation is the pathological basis of these proliferative vascular diseases; we therefore applied a well-established neointimal lesion formation model in balloon-injured rat carotid artery. We demonstrated for the first time that multiple miRNAs were aberrantly expressed in the vascular wall after angioplasty. The multiple miRNA dysregulation and the time course changes of these aberrantly expressed miRNAs match the complex process of neointimal lesion formation, in which multiple genes have been dysregulated. Our results indicate that multiple miRNAs are involved in neointimal lesion formation, although their roles may be diverse.
We found that miR-21 was one of the most upregulated miRNAs in the vascular wall after balloon injury. In addition, miR-21 was also overexpressed in cancers.24,36 To determine the potential role of these aberrantly expressed miRNAs in neointimal lesion formation, we therefore selected miR-21 as our experimental target. We demonstrated that inhibition of miR-21 expression via antisense oligonucleotide-mediated miRNA depletion significantly decreased neointima formation after angioplasty. Our results strongly indicate that miR-21 is an important regulator for neointimal hyperplasia.
Neointimal growth is the balance between VSMC proliferation and apoptosis. The increased VSMC proliferation or the relative decreased VSMC apoptosis are responsible for neointimal formation. To further determine the cellular mechanism of miR-21–mediated effect on neointimal lesion formation, we applied cultured cell models for VSMC proliferation and apoptosis. The results suggest that miR-21 is a proproliferative and antiapoptotic regulator for VSMCs. MiR-21 inhibitor 2′OMe-miR-21–mediated cellular effects on proliferation and apoptosis are miR-21 specific and are not mediated by nonspecific toxic effects, because a high dose of control oligonucleotide, 2′OMe-EGFP (100 nmol/L) had no such cellular effects. In addition, cellular viability experiment using MTT method after transfection of 2′OMe-miR-21 revealed no nonspecific toxicity before the onset of apoptosis (Figure 4D). Furthermore, apoptosis at the highest dose of 2′OMe-miR-21 (100 nmol/L) without serum deprivation was only about 12% after 48-hour miR-21 inhibition (Figure 4E). However, high and disproportionate apoptosis under both miR-21 knockdown and serum deprivation (Figure 4) indicates that a synergetic effect of miR-21 knockdown and serum deprivation on apoptosis may exist, although the mechanism is currently not clear.
The antiapoptotic effect is consistent with the effect of miR-21 on glioblastoma cells.36 However, the antiapoptotic effect was not found in HeLa cells.37 The different effects of miR-21 on different cells suggest that the physiological effect of miRNAs may be cell-type dependent.
The mRNA targets of miRNAs are very complex as miRNAs are able to bind to their mRNA targets with either perfect or imperfect complementarity. Thus, one miRNA may have multiple mRNA targets. The detailed mRNA targets responsible for miR-21–mediated effects on VSMC proliferation and apoptosis are currently unclear. Although we found the activity of caspase-3 was increased in 2′OMe-miR-21–treated VSMCs, the effect may not be the direct effect of miR-21, because there is no direct binding site for miR-21 in the 3′ UTR of caspase-3 mRNA.
With the help of the currently available bioinformatics, we propose that PTEN and Bcl-2, two important signal molecules associated with VSMC growth and apoptosis, might be miR-21 targets. Our results from Western blot analysis indicate that PTEN and Bcl-2 are indeed involved in miR-21–mediated cell proliferation and apoptosis. PTEN could be a direct target of miR-21, because miR-21 inhibition upregulates and miR-21 overexpression downregulates its expression. However, the extent of PTEN express changes was much smaller than that we expected. We think that there are 2 possible reasons: First, PTEN is only one of the multiple target genes of miR-21. Thus, downregulation of miR-21 can only partially reduce PTEN expression; Second, mature miRNA-mediated decrease in its target gene expression is also miRNA-associated multiprotein RNA-induced silencing complex (miRISC)–dependent. For example, the binding of mature miRNA with its target mRNA may require miRISC. Therefore, exogenous transfected miR-21 may only partially reduce PTEN expression because of the limited availability of miRISC. To further confirm the involvement of PTEN in miR-21–mediated effects, we have then determined the effect of miR-21 on its downstream signal molecule Akt using both loss-of-function and gain-of-function approaches. The effects of miR-21 on Akt activity are consistent with the expression changes of PTEN.
In contrast to PTEN, miR-21 inhibition decreases and overexpression increases Bcl-2 expression. Although the results suggest that Bcl-2 is involved in miR-21–mediated effects, the molecular mechanism is unclear. We think there are 2 possibilities. First, Bcl-2 might be an indirect target of miR-21 in VSMCs. MiR-21 may suppress expression of a gene(s) that negatively regulates Bcl-2 expression. Another possibility could be that miR-21 might be able to directly affect Bcl-2 expression, but perhaps not via binding to the 3′ UTR.
Finally, we confirmed the cellular mechanisms of miR-21–mediated effect on neointimal lesion formation in vivo in the vascular wall after angioplasty by modulating the levels of miR-21 with miR-21 inhibitor. The roles of other aberrantly expressed miRNAs need to be defined in future studies.
In summary, miRNAs are aberrantly expressed in the vascular wall after angioplasty. The miRNA expression signature and antisense-mediated depletion reveal an essential role of miRNAs in vascular neointimal lesion formation. MiRNAs may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke.
The authors thank Dr Christopher Waters, Leena P. Desai, and Kenneth Chapman for expert assistance with fluorescent images. The authors also thank Dr David Armbruster from the University of Tennessee Health Science Center for editing assistance.
Sources of Funding
This work was supported by a National Institutes of Health Grant HL080133, an American Heart Association Grant 0530106N, and an American Diabetes Association Grant 105JF60 (to C.Z.).
↵*These authors contributed equally to this study.
Original received October 5, 2006; revision received March 29, 2007; accepted April 23, 2007.
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