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(Hypertension. 2005;45:692.)
© 2005 American Heart Association, Inc.

Original Articles

Angiotensin II Type 2 Receptor–Mediated Gene Expression Profiling in Human Coronary Artery Endothelial Cells

From the Department of Physiology and Functional Genomics, College of Medicine and the Evelyn F. and William L. McKnight Brain Institute, University of Florida, Gainesville.

Correspondence to Mohan K. Raizada, Department of Physiology and Functional Genomics, University of Florida, College of Medicine, PO Box 100274, Gainesville, FL 32610. E-mail mraizada{at}phys.med.ufl.edu

	Abstract

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Despite intensive investigation, the molecular mechanism by which the angiotensin II type 2 (AT₂) receptor exerts its cellular and physiological actions remains elusive. In the present study, we have used microarray expression analysis to identify genes whose expression was regulated by this receptor and to determine its cellular consequences. Lentiviral vector was used to express the AT₂ receptor in human coronary artery endothelial cells (HCAECs), followed by analysis of expression profiles. We observed 5224 genes regulated in an AT₂ receptor ligand-independent manner in HCAECs expressing the AT₂ receptor. In addition, 1235 genes were differentially expressed in response to the AT₂ receptor-specific ligand, CGP42112A. Validity of the expression profiles was demonstrated by real-time reverse-transcriptase polymerase chain reaction quantitation of 5 genes. Because some of these genes could be linked to the regulation of extracellular matrix association, we studied the effect of the AT₂ receptor on cell migration. Expression of the AT₂ receptor resulted in a 2-fold inhibition of HCAEC migration. Taken together, these observations demonstrate that the AT₂ receptor regulates expression of genes relevant to cell migration, protein processing, intracellular signaling, and DNA repair in both ligand-dependent and ligand-independent manners.

Key Words: angiotensin • AT₂ receptor • microarray • cell migration

	Introduction

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It is well-established that angiotensin II (Ang II) elicits its cardiovascular effects such as control of blood pressure, hormone secretion, vasoconstriction, and growth promotion by activation of the Ang II type 1 (AT₁) receptor. In contrast, the role of the Ang II type 2 (AT₂) receptor in cardiovascular physiology remains elusive. It has been proposed that the AT₂ receptor plays a critical role in antagonizing the actions of the AT₁ receptor. As a result, the AT₂ receptor has been proposed to be antiproliferative and to stimulate vasodilatory-relevant mechanisms, leading to beneficial outcomes to the cardiovascular system. These conclusions have been derived primarily using transgenic and knockout mice.^1–7 Despite these inconsistent findings,^1–7 it is generally accepted that the AT₂ receptor has a beneficial function in the cardiovascular system. Strong evidence for this conclusion has recently been provided by our studies.^8,9 They demonstrate that overexpression of the AT₂ receptor in the heart after natural embryonic development prevents the development of cardiac hypertrophy and myocardial fibrosis associated with hypertension.^8,9

Despite limited success in delineating the physiological role, the molecular and cellular actions of the AT₂ receptor remain undefined. This, in part, is caused by a lack of a cardiovascularly relevant in vitro cell system that expresses significant levels of the AT₂ receptor. To overcome this inherent problem, we used a lentiviral vector system to achieve significant expression of the AT₂ receptor in human coronary artery endothelial cells (HCAECs). Using this system, the goal of this study was to use expression profiling to identify genes influenced by the AT₂ receptor that are linked with endothelial functions and to test the involvement of the AT₂ receptor in cellular actions such as endothelial cell migration.

	Materials and Methods

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Lentiviral Construction and Preparation
Lentiviral vectors were constructed, prepared, and titrated as previously described.¹⁰ A lentiviral vector containing green fluorescent protein (Lenti-GFP) was first used to determine the transduction efficiency of the lentivirus in HCAEC. A lentiviral vector that expresses the AT₂ receptor was constructed as previously described.^8,9 This lentiviral vector was created to bicistronically express both the AT₂ receptor and the neomycin resistance gene (Lenti-AT2R-I-Neo) through the use of an internal ribosome entry site. Finally, a control vector was created that only contains the internal ribosome entry site and neomycin resistance genes (Lenti-I-Neo).

Cell Culture
Frozen vials of HCAEC (passage 2) were purchased from Clonetics, thawed, and grown in EGM2-MV growth medium according to the manufacturer’s protocols. Once the cells reached confluence, they were passaged into 4 100-mm diameter dishes at a concentration of 2500 cells/cm². The next day, the cells were transduced with lentiviral vectors at a multiplicity of infection (MOI) of 10 for Lenti-GFP and a MOI of 1 for Lenti-AT2R-I-Neo and Lenti-I-Neo for 20 hours. Lenti-GFP transduced cells were monitored for transduction 48 hours after infection. Cells transduced with Lenti-AT2R-I-Neo and Lenti-I-Neo were selected for neomycin resistance using 800 µg/uL geneticin (G418) in the growth medium for 2 weeks, providing fresh selection media every 2 days. After this selection period, the cells were transferred to 100-mm diameter dishes at a concentration of 2500 cells/cm² to be used for the microarray experiment. After 5 days of growth, HCAECs were treated for 24 hours with 10 nM of the AT₂ receptor-specific agonist, CGP42112A (Sigma), or its resuspension buffer (PBS) as a control (untreated). Finally, total RNA was isolated from the cells using an RNaqueous –4-PCR kit (Ambion) according to the manufacturer’s protocol. In addition, duplicate dishes were used for binding and validation. All experiments were performed with HCAECs of 4 to 6 passages.

Microarray and Analysis
Copy RNA probes were prepared for hybridization to the human U133A microarray chip (Affymetrix) using the protocols described in Affymetrix Gene Chip Expression Analysis Overview. The prepared and fragmented copy RNA (20 µg) was then hybridized to the microarray chip for 16 hours at 45°C. Experiments were performed in duplicate and each sample was hybridized to 1 chip. Thus, data from each group represent mean from 2 chips. This was followed by a series of washing, staining, and scanning according to the manufacturer’s recommendations.

Probe Profiler (Corimbia, Inc) was used to standardize the arrays. Using this program, artifact detection, saturation correction, and outlier detection and removal were performed. Next, the program scaled the mean array intensity of each chip to target intensity (global normalization). Finally, a gene expression value (e-score) was generated, which reflects the expression level of each gene. Genes with an e-score of 25 in at least 1 treatment group were analyzed using a custom software program written in Java using R for 2-way ANOVA. Genes that had a significant change in expression (P<0.05) were analyzed further using Gene Spring software (Silicon Genetics) and gene ontology was determined using NetAffx batch queries (Affymetrix).

Real-Time Reverse-Transcriptase Polymerase Chain Reaction
Primers and probe used to detect the levels of AT₂ receptor was used as previously described.⁸ Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for gene validation was performed using predesigned primers and probes from Applied Biosystems Assays-On-Demand (Catalog numbers: Ubiquitin thiolesterase, Hs00188233_m1; RGS-7, Hs00175619_m1; insulin-like growth factor-binding protein 3, Hs00426287_m1; fibrillin 2, Hs00417208_m1; integrin ß-like 1, Hs00191224_m1). For these experiments, a 2-step real-time RT-PCR protocol was used according to the manufacturer’s protocols. All experiments were normalized to ribosomal 18S as an endogenous control. Quantitation and analysis of gene expression was determined using the comparative cycle threshold (CT) method as described in Applied Biosystems User Bulletin #2.

Ligand Binding Assay
Ligand-specific binding of the AT₂ receptor to ¹²⁵I-sarcosin 1-isoleucine 8 Ang II (¹²⁵I-SI-Ang II) was measured as previously described.^8,11 Values were normalized to the protein content in each reaction as determined by the methods of Lowry et al.¹²

Migration Assay
Transwell cell migration assays were performed in a blind manner to treatment groups using a 48-well Boyden chemotaxis apparatus (Neuroprobe) as previously described.¹³ Briefly, 8-µm membranes were coated overnight in 100 µg/mL type 1 collagen (Sigma) diluted in 20 mmol/L acetic acid. The next day, the membranes were rinsed in PBS and placed over the lower chambers containing EBM-2 medium supplemented with 0.1% bovine serum albumin and 10 ng/mL vascular endothelial growth factor; 5x10³ HCAECs transduced with either Lenti-AT2R-I-Neo or Lenti-I-Neo were added to the upper chamber in medium treated with the AT₂ receptor antagonist, 1 µmol/L PD123 319, 10 nM CGP42112A, or the suspension buffer (PBS). The apparatus was then incubated at 37°C at 5% CO₂ for 3 hours. After incubation, the nonmigrated cells were removed and the migrated cells were stained with Diff-Quick (Fisher Scientific) according to the manufacturer’s protocols. The number of migrated cells was determined by blindly counting 5 randomly chosen fields at 20x magnification.

Statistics
Microarray data were analyzed by 2-way ANOVA as described. All other experiments were analyzed by ANOVA using the Student-Newman-Keuls method for multiple pair-wise comparisons. Results are indicated as mean±SE unless stated otherwise, with statistical significance being set at the 95% confidence interval.

	Results

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Characterization of AT₂ Receptor Transduction in HCAEC
Lenti-GFP was used to determine the efficiency of the lentiviral vector gene transfer system. Initially, Lenti-GFP was used at an MOI of 10 to determine the efficiency of transduction. Figure 1A shows that a majority of the HCAEC showed green fluorescence, indicating GFP expression within 48 hours after transduction.

View larger version (22K): [in this window] [in a new window]   Figure 1. Determination of the efficiency of lentiviral transduction in HCAECs. HCAECs were transduced with Lenti-GFP (MOI 10), Lenti-AT2R-I-Neo (MOI 1), or Lenti-I-Neo (MOI 1) as described in Materials and Methods; 48 hours after transduction, fluorescence microscopy was used to determine the efficiency of lentiviral transduction in HCAECs (A). Real-time RT-PCR analysis was performed using AT2 receptor-specific primers and probes as described in Materials and Methods (B; n=6/group). Scatchard plot and saturation curve analysis was performed on HCAECs transduced with the AT2 receptor in triplicate as described in Materials and Methods (C; n=3/group).

Next, the HCAECs were transduced with either Lenti-AT2R-I-Neo or Lenti-I-Neo, selected for neomyocin resistance, and characterized for AT₂ receptor expression. Cells transduced with the Lenti-AT2R-I-Neo showed 100-fold increase in AT₂ receptor mRNA level compared with HCAECs transduced with the control lentiviral vectors (Figure 1B). This increase in AT₂ receptor mRNA was associated with an increase in AT₂ receptor-specific binding. Although the control transduced cells showed no AT₂ receptor-specific binding, saturation and Scatchard binding analysis revealed that the Lenti-AT2R-I-Neo transduced cells showed a high level of AT₂ receptor-specific binding. The Bmax for the binding was 11.5 pmol/mg of protein with a Kd of 1.25 nM (Figure 1C).

Gene Expression Profiling
Microarray analysis was used as a high-throughput method to determine the molecular effects of AT₂ receptor expression in HCAECs. Of the 33 000 genes represented on the HU133A microarray chip, 5224 genes (Figure 2A) were differentially expressed in a significant manner after AT₂ receptor expression independent of AT₂ receptor ligand. An additional 1235 genes were differentially expressed after treatment with 10 nM CGP42112A for 24 hours (Figure 2B). Genes that were significantly regulated by >70% were analyzed further. This limit was chosen to reduce the number of genes to a more workable number and to focus on those genes whose expression was changed substantially. Using these criteria, we were able to narrow our focus to 35 genes. These genes can be grouped into several general classifications based on gene ontologies. Seven genes are indicated to be important in transcriptional regulation, 6 genes appear to regulate cell maintenance, whereas 3 genes represent a role in signal transduction and in protein processing, and, finally, 2 genes are associated with the extracellular matrix. Table 1 lists the genes that are significantly changed with AT₂ receptor expression independent of ligand, whereas Table 2 lists those genes that were differentially regulated by the addition of the AT₂ receptor-specific agonist, CGP42112A. In addition, genes that were used for validation have been indicated with an asterisk.

View larger version (15K): [in this window] [in a new window]   Figure 2. Scatter plots illustrate the expression patterns of significantly changing genes identified by gene expression profiling. Cells were treated and RNA was prepared as described in Materials and Methods. A, 5224 genes are significantly differentially expressed in the untreated cells transduced with Lenti-I-Neo (Cntrl-Untreat) vs the untreated cells transduced with Lenti-AT2R-I-Neo (AT2R-Untreat). B, In addition, 1235 genes were differentially regulated with the addition of the AT2 receptor-specific agonist, CGP42112A. n=2/group. Genes that were used to validate the microarray experiment are labeled.

Gene Expression Validation
Real-time RT-PCR was used to validate the expression profiling. Genes were chosen based on their possible physiological roles and on the extent to which they were regulated by the AT₂ receptor. Validation of the regulator of G-protein signaling 7 (RGS7) revealed 60% increase in expression in the AT₂ receptor expressing HCAEC (Figure 3A). In addition, ubiquitin thiolesterase (Figure 3B), insulin-like growth factor-binding protein 3 (Figure 3C) and fibrillin 2 (Figure 3D) were all decreased with AT₂ receptor expression by 80%, 55%, and 90%, respectively. These changes reflected changes observed by profiling. However, we found that the expression of integrin ß-like 1 gene was increased by 70% in AT₂ receptor-expressing cells in an AT₂ receptor ligand-independent manner. This was in contrast to the data obtained from the microarrary analysis in which the change in its expression was ligand-dependent when the AT₂ receptor cells were treated with 10 nM CGP42112A. Validation of this observation indicated that the integrin ß-like 1 gene was differentially regulated, but the observed effect was independent of the ligand CGP42112A. There was 70% increase in expression after AT₂ receptor expression independent of ligand (Figure 3E).

View larger version (16K): [in this window] [in a new window]   Figure 3. Real-time RT-PCR validation of AT2 receptor regulation of gene expression. Real-time RT-PCR was performed on mRNA extracted from HCAECs tranduced with either Lenti-AT2R-I-Neo (AT2R) or Lenti-I-Neo (Cntrl) using primers and probes designed for RGS7 (A), ubiquitin thiolesterase (B), insulin-like growth factor-binding protein 3 (IGFBP3) (C), fibrillin 2 (D), and integrin ß-like 1 (E). n=6/group unless noted otherwise. RGS7 and integrin ß-like 1 was performed in 6 per group, but the levels in at least 1 group were below detection and thus could not be analyzed. Fold changes are derived from 2–Ct values.

Role of the AT₂ Receptor in Cell Migration
Because integrin ß-like 1 and fibrillin 2 have previously been linked to endothelial cell association with the extracellular matrix, we hypothesized that AT₂ receptor expression in the HCAECs may be associated with endothelial cell migration. To test this hypothesis, cell migration was assessed using transwell migration assays. We observed 45% decrease in vascular endothelial growth factor-induced cell migration with AT₂ receptor expression that was independent of ligand (Figure 4). In addition, CGP42112A treatment in the HCAECs expressing the AT₂ receptor decreased vascular endothelial growth factor-induced cell migration. These levels were comparable to that of untreated cells despite a modest increase caused by the CGP42112A treatment in the control cells. PD123 319 only partially blocked these AT₂ receptor effects (Figure 4).

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of AT2 receptor expression on migration of HCAECs. Selected HCAEC transduced with either Lenti-AT2R-I-Neo (AT2 receptor) or Lenti-I-Neo (Cntrl) was used in transwell migration assays as described in Materials and Methods. Values represent the average number of migrated cells that were blindly counted from 5 random fields at 20x magnification. n=4 to 6/group and were repeated 3 times with similar results. *P<0.05 versus its control, P<0.05 versus untreated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The most significant finding of this study is that it demonstrates that the AT₂ receptor regulates a class of genes whose expression seems to be linked to HCAECs migration. This suggests that the AT₂ receptor may be critical in the control of endothelial cell migration and angiogenesis. In addition, our data show that there are numerous genes whose expression is regulated by the AT₂ receptor in a ligand-independent manner. The following supports this view. First, there were 5224 genes significantly regulated by AT₂ receptor independent of ligand. Second, the regulation of integrin ß-like 1 that was shown to be differentially regulated with AT₂ receptor agonists in the microarray study was shown to actually be regulated in a ligand-independent manner during validation experiments. Finally, migration assays showed a significant decrease in cell migration without the addition of ligand. Despite a study by Miura and Karnik¹⁴ that illustrated a role for ligand-independent AT₂ receptor activation in apoptosis, very few AT₂ receptor ligand-independent effects have been described. Our results indicate that the AT₂ receptor may have numerous molecular, cellular, and physiological effects that are independent of ligand activation. A better understanding of these ligand-independent effects will be important in the development of therapeutics inhibiting the renin angiotensin system.

A previous study by Benndorf et al showed that the AT₂ receptor decreased endothelial cell migration and tube formation, thus indicating an important role for this receptor in the regulation of angiogenesis.¹³ In our microarray analysis, we observed differential regulation of integrin ß-like 1 and fibrillin 2. Because both of these genes can be associated with the regulation of endothelial cell attachment to the extracellular matrix, we hypothesized that the AT₂ receptor may regulate endothelial cell migration in our cell system. When we tested this hypothesis using an in vitro transwell cell migration assay, we found that the AT₂ receptor decreases cell migration. In doing so, we were able to associate our microarray results with a physiological function and provide further proof of principle for the other differentially regulated genes found in the microarray study. In addition to the aforementioned genes, other differentially regulated genes identified by our microarray analysis may elucidate novel signaling mechanisms for this physiological effect.

HCAECs were chosen for these experiments for several reasons. HCAECs have previously been shown to express both AT₁ and AT₂ receptors at earlier passages.^15,16 This indicates that all of the necessary machinery is present in these cells to reflect a true effect of AT₂ receptor expression. In addition, AT₂ receptor overexpression did not influence endogenous AT₁ receptor levels. Therefore, using this model, we are able to effectively study the role of the AT₂ receptor without the confounding effects of the AT₁ receptor in a cell type that naturally expressed the AT₂ receptor. In addition, endothelial cells play an important role in vascular physiology and their dysfunction has been linked to many physiological and pathological effects such as angiogenesis, atherosclerosis, and vasoconstriction/vasodilation.

Finally, we were able to elucidate novel AT₂ receptor-mediated regulation of gene transcription that may be translated into different physiological effects. For example, ubiquitin thiolesterase was shown to have decreased expression after AT₂ receptor transduction, indicating that the AT₂ receptor plays a role in decreasing the ubiquitination process. This is especially interesting because Weekes et al have previously reported hyperubiquitination in both dilated cardiomyopathy and ischemia of the heart.¹⁷ Another interesting gene that was differentially regulated by the AT₂ receptor was RGS7. Although there is no direct evidence, it can be speculated that the AT₂ receptor may decrease AT₁ receptor-mediated increases in calcium mobilization through the regulation of this gene.¹⁸

Taken together, these results indicate that the AT₂ receptor regulates expression of only a limited number of genes whose expression are associated with cell migration, protein processing, intracellular signaling, and DNA repair. This indicates that the cellular responses of AT₂ receptor in HCAEC require the expression of only a few genes. This provides us a pathway on which future studies can be based. Further characterization of these AT₂ receptor-regulated genes may prove to be an important task in the elucidation of novel AT₂ receptor physiological processes.

Perspectives
The present study describes the identification of genes whose expression is regulated by the AT₂ receptor. Some of these genes are relevant in the control of vasculogenesis, cancer, and cardiovascular disease. Thus, these results may indicate novel targets for the regulation and treatment of these diseases.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL33610 and HL 56921. Beverly (Metcalfe) Falcón was a predoctoral fellow of the AHA Florida/Puerto Pico Affiliate, and Dr Veerasingham is a postdoctoral associate of the Canadian Institutes of Health Research. The authors thank the University of Florida ICBR Microarray Core Facility for their help with the profiling experiments and analysis software, and Nichole Herring for her secretarial assistance.

	Footnotes

 
Dr Falcón’s current affiliation is the Department of Anatomy and the Cardiovascular Research Institute, University of California San Francisco.

Received October 11, 2004; first decision October 31, 2004; accepted December 14, 2004.

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This Article Abstract Full Text (PDF) All Versions of this Article: 45/4/692    most recent 01.HYP.0000154254.89733.29v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Falcón, B. L. Articles by Raizada, M. K. Search for Related Content PubMed PubMed Citation Articles by Falcón, B. L. Articles by Raizada, M. K. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Related Collections Angiogenesis Functional genomics Gene expression Other Vascular biology