Tobacco smoke dysregulates endothelial vasoregulatory transcripts in vivo
J. Gregory Maresh1,
Huaxia Xu1,
Nan Jiang1,
C. Gary Gairola2 and
Ralph V. Shohet1
1 Department of Internal Medicine-Cardiology, University of Texas Southwestern Medical Center, Dallas, Texas
2 Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky
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ABSTRACT
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We hypothesized that human smoking and its deleterious effects on endothelial function can be modeled by exposure of mice to tobacco smoke, and further that these changes would be reflected in gene regulation in vascular endothelium. We used for these studies a mouse strain that expresses green fluorescent protein under the control of an endothelial-specific promoter, Tie-2. Mice were exposed to sidestream smoke from reference cigarettes at 34 mg total suspended particulates/m3. After exposure for 5 days/wk for 1 and 6 wk, aortas were pooled from treatment and control groups. Endothelial cells were rapidly isolated by collagenase treatment followed by fluorescent activated cell sorting to yield populations of >95% purity. RNA isolated from >500 endothelial cells was amplified and analyzed on deeply representative long oligo microarrays. Transcripts dysregulated by >2.5-fold were confirmed by real-time PCR and selected proteins by immunofluorescent localization. In the endothelial cells, the observed more than threefold upregulation of complement factor H (Cfh), calcitonin receptor-like (Calcr1), and soluble epoxide hydrolase (Epxh2) may play a role in hypertensive responses of the vasculature to smoking. We have identified gene regulation in vivo in vascular endothelium that potentially underlies hypertensive responses to tobacco smoke.
environmental tobacco smoke; microarray; epoxide hydrolase
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INTRODUCTION
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CIGARETTE SMOKE EXPOSURE is a principal cardiovascular risk factor contributing to atherosclerosis(16) and hypertension(4). Even environmental "second-hand" tobacco smoke conveys significant risk of increased cardiovascular events on chronic exposure (11). Endothelial dysfunction is a readily observed feature of the human vascular response to environmental tobacco smoke that has been studied in passive smokers (2) and occupationally exposed workers (28). For example, a single 30-min exposure to environmental tobacco smoke can cause coronary endothelial dysfunction in humans (24).
Elucidating endothelial transcriptional response to environmental tobacco smoke is one way to understand the vascular effects of this environmental pollutant. Uncovering dysregulated genes will help to identify the specific pathways and mechanisms of smoke-induced endothelial dysfunction and may lead to improved diagnosis and treatment of its repercussions.
Transcriptional alterations have previously been identified within cultured human umbilical vein endothelial cells exposed to smoke extract, revealing changes in the abundance of mRNAs from genes involved in inflammation, matrix degradation, and proliferation (22). Also, exposure of cultured endothelial cells to nicotine has been performed and analyzed with gene arrays (29). However, the levels and kinds of smoke components administered to monocultured endothelium during in vitro exposure may not represent those that occur in vivo, especially when the filtration capacity of the respiratory tract and detoxifying ability of the organismal anti-oxidant system and liver are considered. Moreover, the endothelial response to these toxins may not be the same in cultured cells that have been modified by culture conditions and lack the paracrine and endocrine effects expected in vivo. Therefore, an evaluation of the in vivo transcriptional response to environmental tobacco smoke is necessary to more fully understand the adaptive and pathological responses of endothelium to this toxin.
Here we report the use of a sidestream cigarette smoke exposure model that, as previously demonstrated, can accelerate atherosclerosis (9). Combining this model with techniques to analyze in vivo endothelial transcriptional responses(20), we have identified novel transcriptional effects of tobacco smoke within the aortic endothelium.
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METHODS AND MATERIALS
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Animals.
Mice homozygous for the Tie2-green fluorescent protein (GFP) transgene [Tg(TIE2GFP)287Sato; stock no. 003658] were obtained from Jackson Laboratories (Bar Harbor, ME) and bred for these experiments. Males were used at 816 wk of age. Controls were siblings of the treated animals. All procedures were approved by the Institutional Animal Care Committees of University of Texas (UT) Southwestern and the University of Kentucky.
Exposure.
Mice were exposed to sidestream cigarette smoke as previously described. This system uses the University of Kentucky reference cigarettes, 2R4F, burned in a Heiner-Borgwald device to generate a sidestream cigarette smoke atmosphere for whole body exposure of unrestrained animals in a Hinners chamber adjusted to 34 mg particulates/m3 with the addition of HEPA-filtered air. Groups of eight mice were exposed for 4 h/day, 5 days/wk, for 1 and 6 wk. This procedure for smoke-exposure has been shown to generate 75150 ng/ml plasma cotinine and 89% carboxyhemoglobin (9). As controls, groups of age-matched mice were exposed to HEPA-filtered air in the same apparatus. After completion of the last exposure session, smoke-exposed and control mice were shipped from the University of Kentucky to UT Southwestern overnight and killed and processed within 24 h of the final exposure period. Two independent groups of experimental and control animals were treated and analyzed after 1 and 6 wk of exposure.
Cell isolation.
Animals were euthanized by CO2 asphyxiation and cervical dislocation. The aortas from the iliac bifurcation to the aortic root were excised by dissection and freed of any adherent tissue. The luminal blood was removed, and the aortas were sliced into 2-mm segments. The aortic segments pooled from 34 animals were suspended in 5 ml of Dulbeccos PBS with 2 mg/ml dextrose. The suspension was combined with 5 ml of prewarmed PBS containing 5 mg/ml type II collagenase (Worthington) and deoxyribonuclease (300 U), agitated continuously at 37°C on a shaking platform, and triturated 10 times every 10 min for a total digestion period of 4060 min to generate a single cell suspension. The cell suspension was maintained at 04°C throughout the remainder of the isolation, which lasted 23 h total. The suspension was combined with 10 ml of 10% FBS in DMEM, and cells were collected by centrifugation and resuspended in 10 ml of PBS. This suspension was then filtered through a sterile 40-µm mesh filter to remove undigested tissue fragments. After centrifugation, the pellet was resuspended in 3.5 ml of PBS containing 0.5 mM EDTA, 30 U/ml DNase I, 3% FBS, and 2 mg/ml dextrose. The suspension was once again filtered through a 40-µm mesh filter.
Aortic cell suspensions were sorted using a MoFlo from Dako Cytomation (Carpinteria, CA). Cells were excited by a 488-nm laser, and GFP signals were collected via the FL1 channel (510550 nm). A pressure of up to 30 PSI was employed, generating 5,00010,000 events/s. Positive cells (500120,000) were collected directly into 2 ml of TRIzol (Invitrogen).
RNA was extracted according to the manufacturers protocol, with the addition of 10 µg of glycogen added at the RNA precipitation step. One-third of the RNA isolated from cells was then subjected to two cycles of amplification, as described (10), using the Message Amp kit (Ambion, Austin, TX) according to manufacturers instructions. This produced
30 µg of amplified cRNA after two rounds of amplification. Also, a similar amount of the endothelial RNA was amplified into cDNA in a single cycle with the Ovation system (Nugen, San Carlos, CA), used according to the manufacturers instructions.
It is difficult to assess the integrity of the tiny amounts of RNA obtained from fluorescence-activated cell sorting (FACS)-sorted cells, (no more than 300 ng from the best sort). However, after two rounds of amplification, the cRNA product ultimately generated was considered to be of acceptable molecular size range as determined by agarose gel electrophoresis with ethidium bromide staining (a generally homogeneous smear ranging from 200 bp to 2 kbp).
Probe preparation and array hybridization.
Two cycles of linear RNA amplification generated adequate probes for array comparisons. Amplified control and smoke-exposed cRNA (2 µg) was directly labeled with Cy3 and Cy5 with the Micromax ASAP RNA Labeling kit (Perkin Elmer) according to the manufacturers instructions and hybridized overnight to spotted glass oligonucleotide microarrays with two copies of the 13,000 gene murine-long oligo V1 (set 1 and 2; Qiagen, Valencia, CA). Scanning was performed with a Genepix instrument (Axon Instruments, Union City, CA), and Gene Traffic software (Iobion, La Jolla, CA) was used to analyze the data. Average 1- and 6-wk fold changes of microarray features (normalized according to the Lowess subgrid method) were calculated from triplicate hybridizations, which included a single dye reversal.
Real-time PCR confirmation.
We designed oligonucleotide amplimers from the cDNA sequences that were predicted to cross an intron; if the mouse gene organization was not available, we used homology to the human gene. Sequences are available in Table 1. All primer sets were assessed by gel electrophoresis to confirm amplification of a single PCR product of the expected size from endothelial cDNA. These primers were used to amplify product from cDNA representing 5 ng of total RNA. PCR was run in triplicate with SYBR Green fluorophore (Molecular Probes, Portland, OR) in an Opticon device (MJ Research, Waltham, MA). A standard two-phase reaction (95°C 15 s, 60°C 1 min) worked for all amplifications.
The relative expression level for each gene was interpolated from a standard curve generated from a series of cDNA dilutions at cycle times where CT, the threshold intensity, was clearly exceeded. In each real-time PCR run, the abundance of GAPDH and/or cyclophilin A was assessed in parallel, and expression fold changes for genes of interest were calculated by normalization to GAPDH as a loading control. By the microarray analysis, GAPDH mRNA abundance was invariant with 1 or 6 wk of smoke exposure.
The hearts and lungs of smoke-exposed and control mice were collected and rapidly frozen at the time of death, and RNA was extracted from frozen tissues with TRIzol. RNA was pooled from three animals of smoke-exposed or control groups. Real-time PCR analysis of the total heart and lung response to smoke was performed with cDNA derived from 2 µg of pooled RNA.
Immunofluorescent localization.
Two mice exposed to smoke for 6 wk as well as two 6-wk control mice were killed with subsequent rapid perfusion fixation of the heart and aorta with 4% paraformaldehyde in PBS. These tissues were processed for paraffin thin sections, deparaffinized, and visualized by secondary immunofluorescence using sheep anti-Cfh (ABCAM, Cambridge, MA), the rabbit anti-Cpe N-term described by Y. P. Loh (3), and the rabbit anti-Epxh2 described by B. D. Hammock (5), all at 1:250, along with appropriate Cy3-labeled Donkey anti-sheep and anti-rabbit secondary antibodies (Jackson Immunoresearch) at 1:500.
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RESULTS
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Microarray and real-time PCR.
Analysis of experiments that employed the murine V1 microarray hybridized to Cy dye-labeled endothelial cRNA of treated vs. control animals revealed 47 transcripts to be dysregulated by >2.5-fold after either 1 or 6 wk of smoke exposure. The hybridization results are available within the Gene Expression Omnibus database under the accession numbers listed in Table 2. Thirty-six of these regulated genes identified in the microarray analysis were selected for confirmation by real-time PCR. Of these, 33 (92%) exhibited similar regulation by both methods. These data are graphically presented in Fig. 1. Seventeen of these genes exhibited >2.7-fold dysregulation at both of the smoke exposure times.

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Fig. 1. Transcriptional responses determined by microarray and subsequently confirmed by real-time PCR, expressed as natural log of fold change on smoke exposure. A: genes with >2.5-fold dysregulation on either 1 or 6 wk of smoke exposure. B: the real-time PCR determination of these transcripts.
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Of the genes presented in Fig. 1, Cfh, Epxh2, Cpe, and Calcrl were considered of special interest because of their potential roles in vasoregulation, and were examined in the lung and heart of mice exposed to environmental smoke for 6 wk. These real-time PCR results are presented in Fig. 2, indicating upregulation of Epxh2, Cfh, and Cpe by >2.7-fold within the smoke-exposed lung, with lower regulation in the heart. Cardiac Cpe levels were below the threshold level of detection (results not shown).

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Fig. 2. Transcriptional responses in whole heart and lung measured using real-time PCR. Bar graph represents natural log of fold regulation in Epxh2, Cfh, Calcrl, and Cpe from mice exposed 6 wk to environmental tobacco smoke.
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To confirm the findings shown in Fig. 1, we examined the aortic expression of Epxh2, Cfh, and Cpe, utilizing immunofluorescent localization to discern any changes occurring with environmental smoke exposure of 6 wk. Immunofluorescent localization with antiserum directed against Epxh2, presented in Fig. 3, reveals upregulation of this protein after 6 wk of environmental smoke exposure. In results not shown, immunofluorescent localization studies of Cpe revealed its upregulation within the endothelium of the capillary bed of the periaortic fat but not within the aortic luminal endothelium. Also in results not shown, Cfh (a major secreted plasma protein), although strongly localized to endothelium by this method, was not apparently upregulated in the aortic lumen after 6 wk of smoke exposure.

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Fig. 3. Immunofluorescent localization of Epxh2 within the aorta after 6 wk of environmental smoke exposure vs. control is revealed by Cy3 localization. Representative fields of an experimental and control sample are shown.
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To further confirm the endothelial transcriptional responses to environmental smoke and to study the biological variability in such responses, exposures of mice were duplicated at both 1 and 6 wk. The second set of samples and the original 1- and 6-wk samples of endothelial RNA were amplified with the Nugen Ovation kit, which generates adequate cDNA for real-time analysis in a single round. Real-time PCR for Cpe, Epxh2, Cfh, and Calcrl was then performed with this cDNA. These results are presented in Fig. 4, revealing consistent and reproducible upregulation of Epxh2 by greater than a single log (2.7-fold). The upregulation of Cpe and Cfh was also concordant with the results presented in Fig. 1. Calcrl was not reproducibly detected by real-time PCR within both sets of Nugen-amplified cDNA.

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Fig. 4. Real-time PCR analysis: smoke-induced transcript-level responses of Cpe, Epxh2, and Cfh on 1 and 6 wk of exposure in duplicate experiments (designated I and II). Endothelial cell RNA was amplified via the Nugen SPIA process. Natural log of fold change was determined for each transcript.
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DISCUSSION
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Changes in transcript abundance occurring within the endothelium in vivo during chronic exposure to smoke are likely to reflect the endotheliums autocrine response to numerous smoke components (CO, nicotine, > 4,000 others) as well as to secondary smoke metabolites (lipid metabolites, peroxynitrite, etc.) generated by various nonendothelial cells and tissues. Given the pivotal role of the vascular endothelium in maintaining hemostasis and vascular tone, gene regulation in these cells in response to inhalation of tobacco smoke should implicate mechanisms of both toxicity and host response. Our evaluation of the in vivo endothelial transcriptional response to tobacco smoke does indeed identify some of the underlying toxic manifestations of exposure to environmental tobacco smoke within the vasculature.
The acute vascular responses to tobacco smoke have been ascribed to oxidative stress, which can generate reactive oxygen species (ROS) such as superoxide anion (27) These ROS can react with nitric oxide to form peroxynitrite, an uncoupler of the activity of eNOS(15). Peroxynitrite is also associated with vascular mitochondrial DNA damage and nitration of superoxide dismutase-2 in mice exposed to cigarette smoke at levels comparable with those described here (1, 14). Thus it has been suggested that rapid loss of the endotheliums ability to generate nitric oxide can help explain acute endothelial dysfunction. Here we demonstrate endothelial adaptive responses that accompany the chronic manifestations of smoke exposure; these include hypertension, increased endothelial adhesiveness, increased vascular permeability, and vascular remodeling.
As shown in Fig. 1, a number of endothelial genes undergo dysregulation in response to environmental tobacco smoke. These include some with known or potential relevance to vasoregulation, including Epxh2 (epoxide hydrolase-2, or soluble epoxide hydrolase), Cfh (complement factor H, or adrenomedullin binding protein-1), and Calcrl(calcitonin receptor-like). Epxh2 catalyzes the hydrolysis of the endogenous vasorelaxant epoxyeicosatrienoic acid (also known as endothelial-derived hyperpolarizing factor; Ref. 13), and we consider its upregulation with smoke exposure to suggest an important role in causing or exacerbating smoke exposure-related hypertension. Cfh, in addition to its established role in regulation of complement activation, has been shown to bind to the vasodilator adrenomedullin and modulate its effects (30). Calcrl is part of the receptor complex for adrenomedullin and calcitonin gene-related peptide (12). The finding of two genes highly regulated by smoke exposure, that both impinge on adrenomedulin, strengthens the conclusion that this vasodilatory peptide is involved in vascular responses to smoking. Cpe, or carboxypeptidase E, is a neuroendocrine prohormone convertase with the potential to process atrial natriuretic factor (ANF) (18, 8) and other regulatory peptides. Interestingly, Cpe(/) null mice exhibit obesity and diabetes (7), and we speculate that upregulation of Cpe may be involved in the weight loss associated with smoking. In results not shown, mice exposed to smoke for 1 and 6 wk weighed 12 and 3% less, respectively, than control mice.
The vasoregulatory changes that occur with smoking are complex. Acute effects of tobacco smoke include hypertension, but paradoxically smokers have lower blood pressure in epidemiological surveys (23). Cessation of active smoking by an individual does lower blood pressure (21). Acute experimental exposure to sidestream cigarette smoke increases blood pressure and aortic stiffness in humans (19). Ambulatory blood pressure is higher in children exposed to environmental tobacco smoke (25). We consider dysregulation of vasoregulatory molecules such as Epxh2, Calcrl, and Cfh to be an important aspect of the complex cardiovascular response to chronic environmental smoke exposure.
The most robust finding presented here is that of a threefold increase in Epxh2 on environmental smoke exposure within FACS-sorted aortic endothelium. This finding was confirmed by localized upregulation of Epxh2 protein in the aorta on smoke exposure. Increased Epxh2 levels in response to smoking are consistent with recent published reports of the key role of Epxh2 in vasoregulation. Studies of Epxh2 inhibitors used as a means of decreasing experimental hypertension (13, 17) and studies of gene polymorphisms in rats (6) and humans (26) all indicate that even small alterations in Epxh2 activity may affect vascular tone. We speculate that therapies that inhibit Epxh2 might be particularly efficacious in the treatment of hypertension in patients who smoke.
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: R. V. Shohet, Internal Medicine-Cardiology, U. T. Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390 (E-mail: ralph.shohet{at}utsouthwestern.edu).
10.1152/physiolgenomics.00310. 2004.
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