AJP - Heart Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 294: H1075-H1085, 2008. First published December 21, 2007; doi:10.1152/ajpheart.00913.2007
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures
Right arrow All Versions of this Article:
294/2/H1075    most recent
00913.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rose, P.
Right arrow Articles by Lounsbury, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rose, P.
Right arrow Articles by Lounsbury, K. M.

Genes overexpressed in cerebral arteries following salt-induced hypertensive disease are regulated by angiotensin II, JunB, and CREB

Patricia Rose,1 Jeffrey Bond,2 Scott Tighe,4 Michael J. Toth,3 Theresa L. Wellman,1 Eva Maria Briso de Montiano,1 Martin M. Lewinter,3 and Karen M. Lounsbury1,4

Departments of 1Pharmacology, 2Microbiology and Molecular Genetics, and 3Medicine, and the 4Vermont Cancer Center, University of Vermont, Burlington, Vermont

Submitted 6 August 2007 ; accepted in final form 14 December 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although changes in gene expression are necessary for arterial remodeling during hypertension, the genes altered and their mechanisms of regulation remain uncertain. The goal of this study was to identify cerebral artery genes altered by hypertension and define signaling pathways important in their regulation. Intact cerebral arteries from Dahl salt-sensitive normotensive and hypertensive high-salt (HS) rats were examined by immunostaining, revealing an increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and expression of the proliferative marker Ki-67 in arteries from hypertensive animals. Arterial RNA analyzed by microarray and validated with RT-quantitative PCR revealed that jun family member junB and matricellular genes plasminogen activator inhibitor-1 (PAI-1) and osteopontin (OPN) were significantly overexpressed in HS arteries. Fisher exact test and annotation-based gene subsets showed that genes upregulated by Jun and Ca2+/cAMP-response element-binding protein (CREB) were overrepresented. A model of cultured rat cerebrovascular smooth muscle cells was used to test the hypothesis that angiotensin II (ANG II), JunB, and CREB are important in the regulation of genes identified in the rat hypertension model. ANG II induced a transient induction of junB and a delayed induction of PAI-1 and OPN mRNA levels, which were reduced by ERK inhibition with U-0126. Silencing junB using small-interfering RNA reduced mRNA levels of OPN but not PAI-1. The silencing of CREB reduced PAI-1 induction by ANG II but enhanced the transcription of OPN. Together, these results suggest that salt-induced hypertensive disease promotes changes in matricellular genes that are stimulated by ANG II, regulated by ERK, and selectively regulated by JunB and CREB.

microarray; Dahl rat; brain arteries; mitogen-activated protein kinase signaling; transcription factors

HYPERTENSION IS A multifactorial disease, linked to both genetic and environmental origins, that ultimately causes direct alterations on the vasculature including smooth muscle cell proliferation and vascular remodeling (2, 34, 48). Rats selectively bred for hypertension studies have been useful models to study the complexity of human essential hypertension (42). The Dahl salt-sensitive (Dahl S) genetic model of hypertension is a paradigm of low renin hypertension observed in humans. Two genetic defects have been identified in the Dahl S strain including a mutation of the {alpha}1 Na+, K+-ATPase gene, affecting the renal basolateral epithelia throughout the nephron, and a restriction fragment-length polymorphism in the renin gene, affecting the proximal tubule (19, 44).

In response to the Na+ challenge of a high-salt (HS) diet, ANG II is not downregulated in the proximal tubules of Dahl S rats, and the resulting dysregulation of ANG II likely contributes to the pathological findings seen in salt-sensitive hypertensive disease (20, 49). The plasma level of ANG II follows a similar trend in that Dahl S rats do not exhibit a compensatory decrease in plasma ANG II in response to a HS diet (5). In addition, the ANG II type 1 (AT1) receptor is functionally upregulated in Dahl S rats thought to be due to increased receptor sensitivity (57). Hence, the defect in Dahl S ANG II regulation in response to a HS diet is not increased ANG II production but the inability to suppress plasma ANG II and an increased responsiveness of AT1 receptors.

Ultimately, the Dahl S genetic rat strain develops a sustained elevation in systolic blood pressure (SBP) that may exceed 200 mmHg when given a HS diet, with evidence of compensatory hypertrophy and congestive heart failure (22, 24). A loss of cerebral blood flow autoregulation followed by blood-brain barrier disruption and hypertensive encephalopathy has also been observed in these animals (47). As such, this model is most representative of malignant hypertension.

The arterial effects of ANG II are primarily mediated through G protein coupling of AT1 receptors on vascular smooth muscle cells (VSMCs) to phospholipases C, D, and A2 (13, 52). These signals mediate acute vasoconstriction and blood pressure regulation and also regulate VSMC proliferation, oxidant signaling, and extracellular matrix interactions (30). Angiotensin-converting enzyme inhibitors and AT1 receptor blockers are beneficial in the treatment of hypertension not only because they reduce blood pressure and improve endothelial cell function but also because they reverse vascular remodeling (1, 32, 40, 57). ANG II is considered a mitogen because of its ability to stimulate VSMC hypertrophy and hyperplasia and its capacity to activate mitogen-activated protein kinases (MAPKs) such as p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) (3, 6, 38).

Cerebral artery VSMCs from Dahl S hypertensive rats exhibit a depolarized membrane potential and increased cytoplasmic Ca2+, c-fos transcription, and Ca2+/cAMP-response element (CRE)-binding protein (CREB) phosphorylation compared with those from their normotensive resistant (Dahl R) controls (51). In this study, data are presented that support the hypothesis that salt-induced hypertensive disease in Dahl S rats induces the expression of specific ERK- and CREB-regulated genes in cerebral arteries. Microarray analysis was utilized to identify the range of genes affected by hypertensive disease, and these results were complemented by an investigation of ERK activation, cell proliferation, and validation using techniques to quantify mRNA and detect protein expression. ANG II stimulation of cultured cerebrovascular smooth muscle cells (cVSMCs) was further employed to define selective roles for ERK, JunB, and CREB in the signaling mechanisms leading to the upregulation of gene targets identified in the hypertension study.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal protocol, cerebral artery isolation, and cVSMC explants. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and followed protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Vermont. All arteries were obtained through tissue sharing from University of Vermont investigators M. LeWinter, Joseph Brayden, Victor May, and Natalia Gokina (IACUC 06-100AP); thus no animals were euthanized exclusively for this study.

For intact studies, male Dahl S rats, weighing ~200 g and ~6 wk old, were obtained from a colony supported by Merck Pharmaceuticals and maintained by Taconic (Germantown, NY). The rats were divided into two groups, and at 7 wk (time = 0) their diets were changed to either an 8% NaCl (HS) or a 0.4% NaCl low-salt (LS) diet. Animals were not started earlier to prevent rapid morbidity observed by others (12, 43). For animals used in the blood pressure study, SBPs were measured weekly using a tail-cuff plethysmograph. For study group animals, beginning at 6 wk after starting the diet, echocardiography was performed weekly, and animals were euthanized when left ventricular dysfunction was detected as previously described (24). The LS animals were euthanized at times that corresponded approximately with the HS animals (see Table 1). After euthanasia, brains were removed and primary branch resistance arteries projecting from the Circle of Willis were dissected (middle and posterior cerebral and posterior cerebellar) in cold HEPES-buffered solution (HBS) composed of (in mmol/l) 10 HEPES (pH 7.4), 140 NaCl, 6 KCl, 2 CaCl2, 1 MgCl2, and 10 glucose. For immunocytochemistry, isolated arteries were fixed in 4% formaldehyde and processed as described in Cerebral artery immunohistochemistry. For microarray and RT-quantitative (q)PCR, arteries were either snap frozen in liquid N2 and stored at –80°C or submerged in RNAlater (Qiagen, Valencia, CA) and stored at –20°C until processed for RNA extraction.


View this table:
[in this window]
[in a new window]

  		Table 1. Dahl S rat study group

 
To generate cultured cVSMCs, middle and posterior cerebral and cerebellar arteries were isolated from female Sprague-Dawley rats (~12 wk, 200 g), cut into rings, and placed in 60-mm culture dishes containing smooth muscle growth media 2 (SMGM2; Cambrex, Palo Alto, CA). Rings were cultured at 37°C/5% CO2, and VSMCs that migrated within 7 to 10 days were trypsinized and transferred to 100-mm plates. Cells were used between passages 2 and 4. cVSMCs were grown to ~70–75% confluence and serum starved in DMEM containing 0.5% fetal bovine serum, 2 mmol/l L-glutamine, 1,000 U/ml penicillin, and 1 mg/ml streptomycin for 48 h before treatment.

ANG II (used at 100 nmol/l) and U-0126 (used at 10 µmol/l) were purchased from Calbiochem (San Diego, CA). SB-203580 (used at 700 nmol/l) was obtained from Biosource (Camarillo, CA), and cell culture reagents were from GIBCO (Grand Island, NY). All other reagents were obtained from Sigma (St. Louis, MO).

Cerebral artery immunofluorescence. Intact artery immunofluorescence was performed as described previously (37a). Briefly, formaldehyde-fixed intact cerebral arteries, secured to sylgard dishes, were sequentially incubated in blocking solution containing 2% BSA and 0.2% Triton X-100 in PBS (Sigma-Aldrich, St. Louis, MO), followed by rabbit anti-p42/p44-phospho-ERK (anti-pERK1/2; Cell Signaling Technologies, Beverly, MA) diluted 1:250 in blocking solution. Arteries were then incubated with Cy5 goat anti-rabbit IgG (Jackson ImmunoResearch, Westgrove, PA) diluted 1:500 in blocking solution. YOYO-1 (Molecular Probes, Eugene, OR) diluted 1:10,000 and containing 24 U/ml RNase A was used to counterstain the nuclei. Arteries were mounted on microscope slides, and images were captured using a Bio-Rad 1000 laser-scanning confocal microscope at x400 magnification. Fluorescence intensity was quantified using mean pixel intensity analysis as described previously (37a). For nuclear intensities, a mask of 25 nuclei was generated from the YOYO images, and the intensity of pERK1/2 was determined within the mask regions.

Cerebral artery immunohistochemistry. Formaldehyde-fixed arteries were paraffin embedded, cut in 5-µm sections, and applied to microscope slides. Tissue sections were deparaffinized using standard techniques (54) and exposed to antigen retrieval buffer containing 10 mmol/l sodium citrate (pH 6.0) for 15 min at 95°C. Immunohistochemistry was performed using the EnVision plus Dual Link Peroxidase and Substrate systems according to the manufacturer's protocol (DakoCytomation, Carpinteria, CA). Rabbit anti-Ki-67 and rabbit anti-osteopontin (OPN) primary antibodies were obtained from Abcam (Cambridge, MA) and used at 1:50 dilution. Sections were counterstained with Mayer's hematoxylin. Digital images were captured using an Olympus BX50 upright light microscope (Olympus America, Lake Success, NY) with an attached Optronics MagnaFire digital camera and software.

RNA isolation from intact cerebral arteries. Each starting sample (n) consisted of arteries pooled from three brains (~2 mg wet tissue wt) with an average yield of 340 ng total RNA. Total RNA was extracted from intact cerebral arteries that had been stored at –80°C or at –20°C in RNAlater using a modified RNeasy protocol for isolation from animal tissues with a DNase I digestion step (Qiagen). To improve tissue disruption and increase RNA yield, the protocol for tissue homogenization was modified to include ~25 mg of 200-µm silicon carbide abrasive beads (MoBio, Carlsbad, CA) in the homogenizing buffer and use of a Pellet Pestle system (Kontes, Vineland, NJ). RNA was eluted with 25 µl RNAse-free H2O. RNA concentrations were determined using a Nanodrop spectrophotometer, and RNA quality was assessed by an Agilent 2100 bioanalyzer (Agilent, Palo Alto, CA).

Cerebral artery RNA amplification and microarray. Each sample (n) consisted of RNA pooled from three different rats on their respective diets (9 total rats/group). RNA samples (n = 3) per group were analyzed to achieve an estimated statistical power of 0.8. RNA amplification and microarray analysis were performed by the University of Vermont DNA facility. RNA samples were double amplified to generate cRNA from the reverse-transcribed cDNA using the method outlined in the Affymetrix Technical Manual (36). After a second cycle, second-strand cDNA synthesis and cleanup, biotinylated cRNA was prepared by in vitro transcription, fragmented, and stained using a streptavidin-phycoerythrin conjugate before application to Affymetrix GeneChip Rat Expression Set 230A (RAE230A) (Affymetrix, Santa Clara, CA). Biotinylated antistreptavidin antibodies were used to amplify the signal. A total of 10 µg of amplified cRNA was applied to each gene chip.

Cerebral artery real-time RT-qPCR. For validation experiments, cDNA was reverse transcribed from different sample pools of RNA than those used in the array analysis. Reverse transcription of 160 ng total RNA from intact cerebral arteries was carried out using an Omniscript reverse transcriptase, RNase-free DNase kit (Qiagen), with an oligo(dT) primer, according to the manufacturer's protocol. RT-qPCR primers and probes were obtained from Assays-on-Demand kits from Applied Biosystems (Foster City, CA). PCR products were detected by TaqMan qPCR, as previously described (37a, 38). Expression levels of target genes were determined using hypoxanthine-guanine phosphoribosyl transferase (hprt) as the internal standard. All samples were run in duplicate from at least three independent experiments, and the comparative cycle threshold (Ct) method for relative quantity (RQ) value was used to calculate relative mRNA expression among samples.

cVSMC transient transfections with small-interfering RNA. Small-interfering RNAs (siRNAs) were obtained from Dharmacon (Lafayette, CO) and included a nontargeting pool (ON-TARGETplus siCONTROL; Cat. No. D-001810-10-05), a positive control cyclophilin B (CypB; siSTABLE cyclophilin B siRNA), and pools targeting junB or CREB (ON-TARGETplus SMARTpool siRNAs; Cat. No. L-087675-00 and Cat. No. L-092995-00). Transfections were performed following the manufacturer's instructions. Briefly, 100-mm plates of cVSMCs were grown to ~75% confluence in SMGM2 media without antibiotics. Cells were transfected with siRNAs (100 nmol/l) complexed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for 4 h, transferred back to SMGM2 for 24 h, and then placed in serum-starving media for a total of 48 h including treatment time.

cVSMC real-time RT-qPCR. Total RNA was extracted from cultured cVSMCs using the RNeasy PLUS protocol for total RNA isolation from animal cells (Qiagen). cDNA was reverse transcribed from a total of 500 ng total RNA, and qPCR was performed as described for intact arteries in Cerebral artery real-time RT-qPCR. All samples were run in duplicate with at least three independent experiments, and the comparative Ct method for RQ was used to calculate relative mRNA levels among samples.

cVSMC Western blot analysis. After treatments, cell plates were transferred to ice, media was aspirated, and the cells were washed first with cold PBS followed by cold hypotonic lysis buffer (HLB) containing (in mmol/l) 25 Tris (pH 8.0), 2 MgCl2, and 5 KCl. Cells were collected by scraping in 60 µl of cold HLB supplemented with a protease/phosphatase inhibitor cocktail containing 1 mmol/l phenyl-methyl-sulfonamide, 20 µg/ml aprotinin, 4 µg/ml leupeptin, 2 mmol/l Na+ orthovanadate, and 2 mmol/l Na+ pyrophosphate. Cell lysates were homogenized, and DNA was sheared by passing the extract through a 26-gauge needle 20 times. Protein concentration was determined using a Bradford protein assay (Bio-Rad, Hercules, CA), and 10 µg of protein were separated by 10% SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed using antibodies specific to CREB (mouse anti-CREB; Cell Signaling Technologies; 1:1,000), OPN [rabbit polyclonal anti-OPN (AB8448); Abcam; 1:1,000], JunB (rabbit polyclonal anti-JunB; Santa Cruz Biotechnology, Santa Cruz, CA; 1:500), and β-actin (rabbit polyclonal anti-β-actin; Cell Signaling Technologies; 1:1,000). All antibodies were diluted in 3% BSA in Tris-buffered saline/0.1% Tween 20 (TBST) and blocked in 3% nonfat dry milk dissolved in TBST. Binding of primary antibody to nitrocellulose blots was detected with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Santa Cruz Biotechnology; 1:5,000) or HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnology; 1:5,000), followed by chemiluminescence using Lumiglo (Kirkgaard and Perry, Gaithersburg, MD).

Statistical analysis. For immunofluorescence, immunohistochemistry, and RT-qPCR data, statistical significance between two groups was determined using a two-tailed unpaired Student's t-test (significance at P < 0.05). Where multiple comparisons were required, significance was assessed using ANOVA for multiple comparisons (Holm-Sidak method). Results are presented as means ± SE.

For statistical analysis of microarray data, signal intensities were assigned to probes in each sample using Affymetrix software (GeneChip operating software). With the use of BioConductor software (www.bioconductor.org), probe-level expression data was normalized using the qspline method of Workman et al. (55), and the robust multichip average (RMA) expression statistics were calculated for each probe set and sample using the method of Speed and coworkers (4, 21). The complete gene expression omnibus (GEO) data set is available under accession No. GSE5488.

Each probe set was associated with the magnitude of differential expression between LS- and HS-treatment groups (M, the average difference of RMA expression statistics), as well as a sample standard deviation (s), a P value (p) based on the Student's t-test, and degrees of freedom (d). Genes identified as differentially expressed (see RESULTS) were judged statistically significant by two methods. Regions describing the joint distribution of M and –log10p under the null hypothesis were constructed after calculating M and –log10p for each of the 10 distinct permutations of the sample labels and contouring a two-dimensional histogram.

Regions describing the joint distribution of M and standard deviation were obtained by explicitly estimating the probability density of the standard deviation, p({sigma}), using an inverted gamma distribution. The probability of obtaining a differential expression statistic greater than or equal to the observed value, conditional upon the estimated standard deviation s, is

Formula
where

Formula


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Dahl S animal study group. The Dahl S model of hypertensive disease is characterized by elevated blood pressure and the development of heart failure in response to a HS diet (22, 24). SBP measurements in Dahl S animals confirmed an early onset and sustained increase in blood pressure in animals fed a 8% NaCl diet (HS) (supplementary Fig. 1; all supplementary material can be found with the online version of this article), and previous experiments found no significant difference in blood pressures between 6 and 12 wk on the HS diet (24). Animals in the study group were evaluated by echocardiography, and HS animals with their LS controls were euthanized when left ventricular dysfunction was detected, after an average of 11 wk on the HS diet. As shown in Table 1, when compared with LS controls, animals receiving a HS diet exhibited a significant decrease in body weight and an increase in lung-body weight ratio, indicating the development of heart failure as a result of malignant hypertension.

Cerebral arteries from salt-induced hypertensive rats have significantly higher ERK activity than LS controls. To determine whether hypertensive disease is associated with an upregulation of cerebral artery ERK activity, cerebral arteries from HS and LS animals were dissected and analyzed by immunofluorescence to detect pERK1/2. As represented in Fig. 1, arteries from HS animals exhibited a significantly elevated level of pERK in both the nucleus and cytoplasm when compared with those from LS controls. Based on the known involvement of ERK in proliferation and remodeling, these data support a role for ERK in the altered signaling pathways that result from chronic hypertension.


Figure 1
View larger version (51K):
[in this window]
[in a new window]

  		Fig. 1. Dahl salt-sensitive (Dahl S) rats with hypertensive disease have increased phospho (p) ERK in intact cerebral arteries. A: middle cerebral arteries were isolated from animals fed a low-salt (LS) or high-salt (HS) diet and then analyzed by whole artery immunofluorescence to detect pERK1/2 (red). YOYO was used to stain nuclei (green). Colocalization (overlay) of pERK1/2 and YOYO appears white. Images were collected by confocal fluorescence microscopy as described in METHODS. Bar = 100 µm. B: quantification of pERK1/2 immunofluorescence was measured by mean pixel intensity over equal areas (total) or within masked nuclear regions (nuclear). *P < 0.05 by Student's t-test; n = 4 experiments.

 
cVSMCs of HS chronically hypertensive rats show marked proliferation compared with those of LS normotensive controls. The impact of hypertensive disease on VSMC proliferation in cerebral arteries was measured directly using immunohistochemical detection of the nuclear proliferation marker Ki-67 (16) and by quantifying nuclei/arterial section. Arteries from HS animals displayed a significantly higher percentage of Ki-67-positive nuclei in both the smooth muscle and endothelial cell layers (Fig. 2). These data suggest that the elevated ERK activity corresponds with increased arterial cell proliferation in cerebral arteries from hypertensive animals.


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 2. Cerebral arteries from Dahl S rats with hypertensive disease have increased smooth muscle cell proliferation. A: middle and posterior cerebellar arteries were isolated from animals fed either a LS or HS diet, and then cross sections were analyzed by immunohistochemistry to detect the proliferation marker Ki-67 (brown nuclear stain). Left: staining in the absence of primary antibody (No 1° Ab). Middle and right: Ki-67-specific staining. Images at left and middle were collected using a x20 objective, and images at right were collected using a x60 objective. B: Ki-67-positive nuclei graphically depicted as a percentage of total nuclei ± SE. **P < 0.005 by Student's t-test; n = 4 animals/group, 2 to 3 arteries/animal. VSMCs, vascular smooth muscle cells; Endo, endothelial cells.

 
Genes important for tissue remodeling are upregulated in cerebral arteries from hypertensive animals. The effect of hypertensive disease on cerebral artery gene expression was determined by microarray analysis using an Affymetrix RAE230A GeneChip array. Of the 15,923 genes represented on the microarray, 133 exhibited a significant 1.5-fold or greater change in expression (99 genes were upregulated and 34 were downregulated in HS compared with LS rats; GEO accession No. GSE5488). Rather than rank genes independently based on the log ratio change (M) or the P value (p), differential expression statistics were represented as a joint distribution using two-dimensional histograms represented by either a volcano plot [log2(M) vs. –log10(p), Fig. 3A] or log2(M) vs. standard deviation (Fig. 3B). This representation of the data allowed the assignment of volcano plot-motivated statistics to each probe set (pv). Contours of pv that exclude 10% and 1% of the probe set were then used to define differential expression-based binary partitions. The pv threshold was supplemented with the condition |log2M| >0.5 and P < 0.2 to eliminate small values from the analysis.


Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 3. Identification of genes of interest using database subsets. A: a volcano plot was created expressing –log10 of the P value (p) on the y-axis versus the log2 fold change (M) on the x-axis to reveal genes of interest in the upper quadrants for the complete probe set. Contours represent the probability density to highlight 10% (inner contour) and 1% (outer contour) of the probe sets. Genes of interest are identified and designated by colored circles: periostin (Postn; red), plasminogen activator inhibitor-1 (PAI-1; green), osteopontin (OPN; blue), and JunB (orange). B: regions describing the joint distribution of log2 fold change (M) and standard deviation (STDV) were obtained by explicitly estimating the probability density of the STDV. Contour lines establish a boundary to highlight the 1% outer contour, and genes of interest are identified as in A (see METHODS for details). C: a volcano plot using the contours from A was overlaid with a subset from the rat genome database of genes associated with hypertension (red), Jun (blue), and Ca2+/cAMP-response element binding protein (CREB; green). Genes identified by the cluster analysis are indicated. D: a probability density plot using the contours from B was overlaid with the subsets described in C. ApoE, apolipoprotein E.

 
Standouts in the analysis of the array data were several genes related to extracellular matrix and tissue remodeling including OPN (Spp1), plasminogen activator inhibitor-1 (PAI-1) (Serpine1), bone-expressed sequence tag 5, and periostin (Fig. 3, A and B, and Table 2). Of note was the finding that guanylyl cyclase (Guacy1a3), a major substrate for nitric oxide-mediated vasodilation, was downregulated in the cerebral arteries from HS rats. Several other forms of Guacyla3 were also significantly reduced, although to a lesser degree.


View this table:
[in this window]
[in a new window]

  		Table 2. Microarray analysis using Affymetrix Rat Expression Set 230A to compare gene transcripts between LS and HS Dahl S rats

 
Fisher exact test was used to examine the hypothesis that genes specified by the transcriptional regulatory element and rat genome databases as related to hypertension and selected transcription factors are upregulated in arteries from Dahl HS animals (Fig. 3, C and D). Differential expression-based partitions of the probe sets at 1% and 10% revealed an overrepresentation of Jun-regulated genes and changes in CREB-regulated genes that approached significance (Table 3). CREB- and Jun-regulated genes overlapped for 10 probe sets, and hypertension- and Jun-regulated genes overlapped for two probe sets. With the use of the hypertension data set, the resulting volcano plot revealed an increase in apolipoprotein E (ApoE) that was not initially significant when analyzed among the entire data set, providing a potential link to genes involved in the pathological course of hypertensive disease.


View this table:
[in this window]
[in a new window]

  		Table 3. Association of gene expression statistics using Fisher exact test

 
Seven genes were selected for validation and further study based on their regulation by ERK signaling and evidence for their transcriptional regulation by ANG II (7). Of these, three were confirmed by RT-qPCR (OPN, PAI-1, and junB), and the reduction in Guacyla3 approached significance (Table 4). Although the other selected genes were not validated, the trends in the data suggest that variability between samples precluded significance. The confidence in the changes associated with genes that did validate is strengthened by the fact that RT-qPCR was performed using RNA sample pools that were distinct from samples used in the array analysis.


View this table:
[in this window]
[in a new window]

  		Table 4. Validation of array results for selected genes altered in cerebral arteries of Dahl S hypertensive rats using RT-quantitative PCR

 
OPN protein expression is increased in cerebral arteries from hypertensive animals. The confirmed extent of induction of OPN mRNA levels in response to hypertensive disease was particularly intriguing due to the previous implications that OPN regulates VSMC proliferation and migration and is overexpressed during postangioplasty restenosis (15, 35, 50). In addition, OPN is a matricellular gene that is known to be transcriptionally regulated by ANG II and ERK signaling (7). To determine whether the induction of OPN mRNA levels correlated with an increase in OPN protein expression, immunohistochemistry was used to detect OPN in cerebral artery cross sections from HS and LS animals. Arteries from HS animals exhibited a significantly higher level of OPN protein staining throughout the cytoplasm of the smooth muscle cell layers (Fig. 4), suggesting that the induction of OPN at the mRNA level is translated to an induction of protein expression. Together, these data suggest that hypertensive disease affects cerebral arteries through enhanced ERK signaling and smooth muscle cell proliferation, which correlates with the induction of matricellular/proliferation-related genes, particularly OPN.


Figure 4
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 4. OPN protein expression is elevated in Dahl S rats with hypertensive disease. Middle cerebral and posterior cerebellar arteries were isolated from animals fed either a LS or HS diet, and then cross sections were analyzed by immunohistochemistry to detect OPN. A: OPN (brown cytoplasmic stain) detected in a representative arterial section from a LS- and HS animal. B: histogram of relative OPN staining intensity ±SE from experiments shown in A and quantified using MetaMorph software. *P < 0.05 by Student's t-test; n = 4 animals/group, 2 to 3 arteries/animal.

 
ANG II exposure of cultured cVSMCs causes induction of hypertension target genes. The genes of focus, junB, PAI-1, and OPN, have been linked to ERK activation in other systems and have inducible activator protein-1 (AP-1) and/or CRE sites within their promoters. In addition, ANG II signaling has been linked to pathologies in the Dahl S hypertensive model and to ERK activation. A culture model of low-passage explanted cVSMCs was thus utilized to define the importance of ERK, JunB induction, and CREB activation in the regulation of junB, PAI-1, and OPN expression. These cells express the VSMC-specific marker calponin and exhibit the ANG II-mediated activation of ERK and CREB, as previously shown by our group and others in other cultured VSMC models (supplementary Fig. 2) (27, 38). A time course of ANG II treatment established an early transient induction of junB mRNA level and protein expression in cVSMCs (Fig. 5A). A delayed induction of PAI-1 and OPN by ANG II was also observed, with significant increases in mRNA levels at 3 and 6 h (Fig. 5B). Of note, the induction of the OPN mRNA level was less than that observed in the Dahl S hypertensive model, likely reflective of higher basal OPN in the cVSMC model. Overall, these data show that ANG II signaling leads to the induction of the same target genes that are upregulated in the Dahl hypertensive model and that JunB upregulation by ANG II precedes the elevation of PAI-1 and OPN mRNA levels.


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 5. ANG II induces transient expression of JunB and delayed expression of PAI-1 and OPN in cerebrovascular VSMCs (cVSMCs). A: cVSMCs were treated with 100 nM ANG II for the indicated times, and mRNA levels for junB were determined by RT-quantitative (q)PCR. Inset: representative Western blot showing time course for JunB protein expression following 100 nM ANG II treatments. Blots were probed for β-actin to confirm equal protein loading. B: cells were treated as in A, and mRNA levels for junB, PAI-1, and OPN were determined by RT-qPCR. RNA concentration was corrected using hypoxanthine-guanine phosphoribosyl transferase (hprt) as the endogenous control, and values were normalized to time 0. Results are expressed as mean relative quantity (RQ) values ± SE (n = 4–7). Symbols denote significance (P < 0.05) by ANOVA when compared with time 0 for *junB, {phi}PAI-1, and #OPN, respectively.

 
ANG II induction of junB, PAI-1, and OPN mRNA levels is reduced by MAPK inhibition. To determine a specific role for MAPKs in the ANG II-mediated response on gene targets, cVSMCs were pretreated with the selective MEK inhibitor U-0126 or the selective p38 inhibitor SB-203580 before ANG II treatments. MEK inhibition reduced the basal expression of junB mRNA and caused a significant inhibition of ANG II induction of both junB and PAI-1 mRNA levels (Fig. 6). In these experiments, ANG II caused an increase, although not significant, in OPN mRNA that was reduced by U-0126 and SB-203580 (Fig. 6C). A closer interpretation of the RT-qPCR data revealed a high basal expression of OPN in the presence of the DMSO vehicle, which has been observed previously in smooth muscle cells (15). Inhibition of p38 resulted in a trend toward reducing the level of ANG II induction of mRNA for all gene targets. MAPKs are thus important in ANG II signaling to gene targets related to hypertension, with a prominent role for ERK.


Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 6. Inhibition of mitogen-activated protein kinases (MAPKs) reduces ANG II transcriptional activation of junB and PAI-1. After 30 min of pretreatment with either DMSO (vehicle control), 10 µmol/l U-0126, or 700 nmol/l SB-203580, cVSMCs were treated with 100 nM ANG II for 30 min (A) or 3 h (B and C), followed by measurement of mRNA for junB (A), PAI-1 (B), or OPN (C) using RT-qPCR. RNA concentration was corrected using hprt as the endogenous control, and values were normalized to the DMSO-untreated control. Histograms represent mean RQ values ± SE. *P < 0.05 by ANOVA compared with DMSO; #P < 0.05 by ANOVA compared with DMSO + ANG II; n = 4 independent experiments.

 
Silencing junB and CREB in cVSMCs using siRNA. To elucidate a role for JunB and CREB in the ANG II transcriptional regulation of gene targets (junB, PAI-1, and OPN), a siRNA transfection technique was developed for cVSMCs. Optimum transfection conditions were first determined using a positive control siRNA, targeting CypB, which achieved 70% knockdown of CypB mRNA (Fig. 7A). These conditions were used to selectively silence junB or CREB, using a nontargeting siRNA (si control) as a control for transfection. As shown in Fig. 7B, the transfection with siRNA targeting junB resulted in a significant 50% reduction in the ANG II-mediated junB transcriptional response at 30 min, which corresponded with a loss of JunB protein expression (Fig. 7C). CREB expression was not affected by ANG II treatment, but transfection with si CREB resulted in a considerable reduction in detectable CREB protein. The si CREB did not significantly inhibit the JunB transcription or its corresponding protein expression, although a slight decrease was consistently observed. Thus efficient and selective knockdown of junB and CREB can be achieved using transfection with siRNAs, and this experimental template was used to explore the effects of junB and CREB knockdown on the regulation of PAI-1 and OPN expression.


Figure 7
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 7. Efficient knockdown of JunB and CREB in cVSMCs using small-interfering (si)RNA. A: cVSMCs were transfected with 100 nmol/l of either nontargeting siRNA pool (si control) or siRNA-targeting cyclophilin B (si CypB). Cells that were not transfected (No Trfx) were used as the endogenous control for transfection. RNA was isolated, and mRNA levels were detected by RT-qPCR using hprt as the endogenous control. Values were normalized to the untreated control. Results are expressed as mean RQ values ± SE. *P < 0.05 by ANOVA compared with si control; n = 4 experiments. B: cells were transfected as in A, with siRNAs targeting junB (si junB) or CREB (si CREB). After treatments with 100 nmol/l ANG II for the indicated times, mRNA levels were detected by RT-qPCR and expressed as in A. *P < 0.05 by ANOVA compared with untreated si control; #P < 0.05 by ANOVA compared with ANG II-treated si control; n = 4–7 independent experiments. C: cVSMCs were transfected and treated with ANG II as in B, followed by analysis of protein extracts by Western blot using antibodies specific to JunB (top), CREB (middle), and β-actin (bottom) as a standard for protein loading. Blots are representative of 3 independent experiments.

 
JunB and CREB have selective roles in the ANG II-mediated regulation of PAI-1 and OPN gene expression. Although silencing of junB transcript had no effect on the induction of PAI-1 by ANG II, silencing CREB transcript prevented the PAI-1 response. CREB is thus important in the ANG II-mediated signaling that results in PAI-1 transcription (Fig. 8A). PAI-1 protein levels were not assessed due to lack of suitable Western blot antibodies. Conversely, si junB significantly reduced both the basal and ANG II-stimulated transcription of OPN, whereas knockdown of CREB resulted in an unexpected induction of OPN mRNA and protein (Fig. 8, B and C). Although both JunB and CREB are regulated through ERK signaling, the data presented here suggest that they have opposing roles related to PAI-1 and OPN gene expression, which may be important with respect to signaling during the development of vascular pathologies as a result of hypertension.


Figure 8
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 8. CREB silencing prevents ANG II induction of PAI-1 mRNA, and junB silencing inhibits OPN expression. cVSMCs were transfected with 100 nmol/l of either si control or si junB or si CREB and then treated with 100 nmol/l ANG II for the times indicated. A and B: PAI-1 and OPN mRNA levels were detected using RT-qPCR, using hprt as the endogenous control and untreated groups as baseline control. Histogram shows results expressed as mean RQ values ± SE. *P < 0.05 by ANOVA compared with untreated si control; #P < 0.05 by ANOVA compared with corresponding ANG II si control; n = 4–7 independent experiments. C: Western blots of cVSMC protein extracts using antibodies specific for OPN and β-actin (loading control). Blots are representative of 5 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Chronic hypertensive disease leads to pathological changes in the arterial wall (1), and the goal of this study was to better define the alterations in signaling pathways and gene expression that underlie these pathologies in cerebral arteries. Here we present the first evidence that salt-induced malignant hypertensive disease in Dahl S animals results in the chronic activation of ERK and increased proliferation of cerebral artery smooth muscle cells. These changes correlate with increased mRNA levels of multiple genes known to be regulated by ERK signaling, most notably the AP-1 transcript junB and the matricellular genes OPN and PAI-1. These findings were extended using a culture model of cVSMCs, revealing parallels between genes altered in chronic hypertension and genes responsive to ANG II signaling. Results also establish, for the first time, selective roles for JunB and CREB in the induction of OPN and PAI-1 by ANG II.

ERK has been associated with altered gene expression in several models of vascular disease (7, 38). Our data showing elevated ERK activation in cerebral arteries from Dahl S hypertensive animals corroborate an earlier study in the same model linking the chronic activation of ERK with glomerular injury in Dahl S rats (18). In cultured VSMCs, ERK phosphorylation induced by mechanical injury leads to the overexpression of OPN protein, and ANG II induces PAI-1 expression in a ERK-dependent manner (8, 31). Furthermore, OPN and PAI-1 were identified in a matricellular gene cluster as highly ANG II responsive (7). Our data thus support the findings of others, implicating an important role for altered signaling among ANG II, ERK, and the expression of OPN and PAI-1.

Both OPN and PAI-1 have been previously implicated as participants in vascular remodeling. OPN is a marker of proliferation in rat VSMCs, and its expression in cultured VSMCs correlates with the downregulation of contractile proteins, suggesting a switch from the contractile to the proliferative phenotype (15, 17, 50, 56). OPN plays a role in coronary artery postangioplasty restenosis through its chemotactic properties, where it mediates smooth muscle cell extracellular matrix invasion of the intimal layer (35). Moreover, OPN has been referred to as a "delayed early gene" because of its role in cell proliferation following mitogenic stimulation in arterial smooth muscle cells (15).

PAI-1 is a potent chemotactic molecule and promotes migration through its interactions with the LDL receptor-related protein and is the primary physiological antagonist to both tissue and urokinase plasminogen activators (14). Increased levels of PAI-1 have also been detected in vascular lesions induced by atherosclerosis or balloon catheter injury (11, 46). PAI-1 promotes proliferation and protects arterial smooth muscle cells from apoptosis, thus there is an increased likelihood of hyperplasia following angioplasty when PAI-1 expression is abnormally elevated (9). Our findings extend these conclusions to cerebral arteries, supporting a role for OPN and PAI-1 in the proliferative changes observed in cerebral arteries of hypertensive rats.

Although not observed previously in cerebral arteries, an induction of OPN and PAI-1 has been observed in other models of hypertensive disease. An array analysis to detect changes in genes from the left ventricle of the heart in the spontaneously hypertensive rat (SHR) model found an overexpression of OPN, PAI-1, fibronectin-1, ApoE, and cathepsin K (45). Upregulation of OPN and PAI-1 was also detected in kidneys from stroke-prone SHRs with malignant hypertension (33). PAI-1 is elevated in the plasma of chronically hypertensive patients, and populations expressing the 4G/4G PAI-1 promoter polymorphism, which confers increased basal PAI-1 transcription, exhibit an increased risk of developing arterial hypertension (29). These data suggest that our observed changes in cerebral artery gene expression are not restricted to the etiology of the hypertensive disease or to arterial tissue and that the Dahl model may be useful for deciphering the consequences of hypertension on cerebral artery function in human disease.

In this study, several AP-1 family member transcripts were variably elevated in cerebral arteries from hypertensive animals including c-jun and c-fos; however, junB transcript was consistently upregulated. JunB has been shown to dimerize and transactivate AP-1 genes but can also act as a negative regulator of c-Jun for some genes (10, 37). In cultured VSMCs, JunB has been detected in active AP-1 complexes in cells responding to receptor tyrosine kinase and G protein-coupled receptor agonists, and its induction is related to the production of reactive oxygen species (41). These findings together with our findings that Jun- and CREB-regulated genes are overrepresented in cerebral arteries from hypertensive animals indicate that signaling through AP-1 and CRE promoter elements may be particularly relevant in the arterial remodeling response.

The early onset and transient nature of JunB induction by ANG II in cultured VSMCs is consistent with the activation of other AP-1 family members responding to mitogen stimulation (26). The delayed stable induction of OPN and PAI-1 transcript by ANG II also agrees with previous findings that prolonged ERK leads to their induction (8, 15). Persistent upregulation of junB in malignant hypertension may thus indicate a defect in the regulation of junB that contributes to remodeling and involves OPN and PAI-1 expression.

Our RNA silencing experiments indicate that JunB is important for the ANG II-mediated upregulation of OPN and that CREB is not required for junB induction but acts as a negative regulator of OPN expression. Although the junB promoter contains a half-site CRE, it also contains both AP-1 and serum response element sites that likely compensate for the CRE through their stimulation by ANG II in our model (28, 53). Two AP-1 sites and a CRE have been identified within the OPN promoter, and a mutational analysis suggests that the AP-1 sites are the most critical (23). The CREB findings were at first unexpected since others have found that dominant-negative CREB reduces OPN expression, and CREB has been associated with c-Fos binding to AP-1 sites on the OPN promoter (23). The differences may be agonist specific, and because ANG II initiates transactivation through both CREB and AP-1, our data suggest that not only AP-1 activity predominates in induction of OPN but that CREB activity interferes with the AP-1 induction.

Contrary to the OPN results, silencing of junB had no effect on the ANG II-mediated induction of PAI-1 mRNA levels, whereas silencing of CREB prevented PAI-1 induction. The PAI-1 promoter contains at least two AP-1 sites, and the CREB target database identifies two half-site CREs. AP-1 has been previously shown in fibroblasts to be important in PAI-1 induction through ERK (25), thus the lack of effect of silencing junB in our studies suggests an alternate AP-1 member may be important for PAI-1 induction by ANG II or that the AP-1 effect is cell-type specific. Although implicated by promoter elements, our data are the first to define a specific role for CREB in PAI-1 induction by ANG II. Taken together, our data support a model for gene expression in cerebral arteries following salt-induced hypertensive disease that includes the upregulation of matricellular genes that are universally regulated by ANG II and ERK and selectively regulated by JunB and CREB (Fig. 9).


Figure 9
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 9. Proposed model for regulation of gene expression in cerebral arteries responding to hypertension. Dysregulated ANG II signaling activates a signaling cascade that includes activation of ERK and selective regulation of gene expression through JunB and CREB.

 
The question arises as to whether the upregulated genes in the hypertensive model contribute to the development of arterial remodeling or are a compensatory healing response to arterial injury. The genes pursued in our study have been linked to both induction of hypertrophy and hyperplasia as well as to healing responses. One possibility is that the vessel wall becomes locked into an endless cycle of mechanical, oxidative, and inflammatory stresses, all linked to compensatory proliferation. Defining a specific role for the genes identified in each of these processes will be facilitated using selective knockdown models to dissect the role of these genes in the context of a hypertensive environment.

This study is the first to use microarray analysis to examine gene expression in intact cerebral vessels from Dahl S malignant hypertensive animals. Our results substantiate previous data and offer new findings related to changes in gene expression induced by hypertension. These data also illustrate how array analyses, especially when evaluated using the Fisher exact test, can be useful tools for assessing sets of genes altered in disease. Our data using cultured VSMCs to elucidate a role for JunB and CREB in the regulation of PAI-1 and OPN give us greater insight into the molecular mechanisms that underlie the overexpression of these genes. In addition to revealing a role for JunB and CREB in the development of hypertensive disease, these results will serve as a springboard of information to explore other sets of genes that contribute to the manifestations of cardiovascular disease in the Dahl S and other models of hypertension.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were primarily supported by National Heart, Lung, and Blood Institute (NHLBI) Grant 1-R01-HL-67351 with additional funding from the Totman Medical Research Trust Fund. P. Rose was supported by NHLBI Minority Supplement Grant 1-R01-HL-67351-S1. M. M. Lewinter was supported by NHLBI Grant R01-HL-52087. Microarray experiments were subsidized by the Vermont Genetics Network through Grant P20-RR16462 from the Institutional Development Award Networks of Biomedical Research Excellence Program of the NCRR.


   ACKNOWLEDGMENTS
 
We thank Timothy Hunter of the DNA facility, Marilyn Wadsworth of the University of Vermont imaging facility, and Daniel M. Roy for assistance in generating and analyzing data. These results are solely the responsibility of the authors and do not necessarily represent the official views of the National Center for Research Resources (NCRR) or National Institutes of Health.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. M. Lounsbury, Dept. of Pharmacology, Univ. of Vermont, 89 Beaumont Ave., Burlington, VT 05405 (e-mail: Karen.lounsbury{at}uvm.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Baumbach GL, Chillon JM. Effects of angiotensin-converting enzyme inhibitors on cerebral vascular structure in chronic hypertension. J Hypertens Suppl 18: S7–S11, 2000.[Medline]
  2. Baumbach GL, Heistad DD. Remodeling of cerebral arterioles in chronic hypertension. Hypertension 13: 968–972, 1989.[Abstract/Free Full Text]
  3. Berk BC, Vekshtein V, Gordon HM, Tsuda T. Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension 13: 305–314, 1989.[Abstract/Free Full Text]
  4. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19: 185–193, 2003.[Abstract/Free Full Text]
  5. Bouhnik J, Richoux JP, Huang H, Savoie F, Baussant T, Alhenc-Gelas F, Corvol P. Hypertension in Dahl salt-sensitive rats: biochemical and immunohistochemical studies. Clin Sci (Lond) 83: 13–22, 1992.[Medline]
  6. Campbell-Boswell M, Robertson AL Jr. Effects of angiotensin II and vasopressin on human smooth muscle cells in vitro. Exp Mol Pathol 35: 265–276, 1981.[CrossRef][Web of Science][Medline]
  7. Campos AH, Zhao Y, Pollman MJ, Gibbons GH. DNA microarray profiling to identify angiotensin-responsive genes in vascular smooth muscle cells: potential mediators of vascular disease. Circ Res 92: 111–118, 2003.[Abstract/Free Full Text]
  8. Chen HC, Feener EP. MEK1,2 response element mediates angiotensin II-stimulated plasminogen activator inhibitor-1 promoter activation. Blood 103: 2636–2644, 2004.[Abstract/Free Full Text]
  9. Chen Y, Budd RC, Kelm RJ Jr, Sobel BE, Schneider DJ. Augmentation of proliferation of vascular smooth muscle cells by plasminogen activator inhibitor type 1. Arterioscler Thromb Vasc Biol 26: 1777–1783, 2006.[Abstract/Free Full Text]
  10. Chiu R, Angel P, Karin M. Jun-B differs in its biological properties from, and is a negative regulator of, c-Jun. Cell 59: 979–986, 1989.[CrossRef][Web of Science][Medline]
  11. Coban E, Ozdogan M. The plasma levels of plasminogen activator inhibitor-1 in subjects with white coat hypertension. Int J Clin Pract 58: 541–544, 2004.[CrossRef][Web of Science][Medline]
  12. Dahl LK, Knudsen KD, Heine MA, Leitl GJ. Effects of chronic excess salt ingestion. Modification of experimental hypertension in the rat by variations in the diet. Circ Res 22: 11–18, 1968.[Abstract/Free Full Text]
  13. De Gasparo M, Whitebread S, Mele M, Motani AS, Whitcombe PJ, Ramjoue HP, Kamber B. Biochemical characterization of two angiotensin II receptor subtypes in the rat. J Cardiovasc Pharmacol 16 Suppl 4: S31–S35, 1990.
  14. Degryse B, Neels JG, Czekay RP, Aertgeerts K, Kamikubo Y, Loskutoff DJ. The low density lipoprotein receptor-related protein is a motogenic receptor for plasminogen activator inhibitor-1. J Biol Chem 279: 22595–22604, 2004.[Abstract/Free Full Text]
  15. Gadeau AP, Campan M, Millet D, Candresse T, Desgranges C. Osteopontin overexpression is associated with arterial smooth muscle cell proliferation in vitro. Arterioscler Thromb 13: 120–125, 1993.[Abstract/Free Full Text]
  16. Gerdes J, Lemke H, Baisch H, Wacker H, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133: 1710–1715, 1984.[Abstract]
  17. Giachelli CM, Bae N, Almeida M, Denhardt DT, Alpers CE, Schwartz SM. Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques. J Clin Invest 92: 1686–1696, 1993.[Web of Science][Medline]
  18. Hamaguchi A, Kim S, Izumi Y, Iwao H. Chronic activation of glomerular mitogen-activated protein kinases in Dahl salt-sensitive rats. J Am Soc Nephrol 11: 39–46, 2000.[Abstract/Free Full Text]
  19. Herrera VL, Ruiz-Opazo N. Alteration of alpha 1 Na+,K+-ATPase 86Rb+ influx by a single amino acid substitution. Science 249: 1023–1026, 1990.[Abstract/Free Full Text]
  20. Hodge G, Ye VZ, Duggan KA. Salt-sensitive hypertension resulting from nitric oxide synthase inhibition is associated with loss of regulation of angiotensin II in the rat. Exp Physiol 87: 1–8, 2002.[Abstract]
  21. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP. Summaries of affymetrix genechip probe level data. Nucleic Acids Res 31: e15, 2003.[Abstract/Free Full Text]
  22. Iwanaga Y, Kihara Y, Hasegawa K, Inagaki K, Yoneda T, Kaburagi S, Araki M, Sasayama S. Cardiac endothelin-1 plays a critical role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive hypertensive rats. Circulation 98: 2065–2073, 1998.[Abstract/Free Full Text]
  23. Jalvy S, Renault MA, Lam Shang Leen L, Belloc I, Reynaud A, Gadeau AP, Desgranges C. CREB mediates UTP-directed arterial smooth muscle cell migration and expression of the chemotactic protein osteopontin via its interaction with activator protein-1 sites. Circ Res 100: 1292–1299, 2007.[Abstract/Free Full Text]
  24. Kameyama T, Chen Z, Bell SP, VanBuren P, Maughan D, LeWinter MM. Mechanoenergetic alterations during the transition from cardiac hypertrophy to failure in Dahl salt-sensitive rats. Circulation 98: 2919–2929, 1998.[Abstract/Free Full Text]
  25. Keeton MR, Curriden SA, van Zonneveld AJ, Loskutoff DJ. Identification of regulatory sequences in the type 1 plasminogen activator inhibitor gene responsive to transforming growth factor beta. J Biol Chem 266: 23048–23052, 1991.[Abstract/Free Full Text]
  26. Kim S, Iwao H. Stress and vascular responses: mitogen-activated protein kinases and activator protein-1 as promising therapeutic targets of vascular remodeling. J Pharm Sci 91: 177–181, 2003.[CrossRef][Web of Science]
  27. Liao DF, Monia B, Dean N, Berk BC. Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. J Biol Chem 272: 6146–6150, 1997.[Abstract/Free Full Text]
  28. Lu D, Yang H, Raizada MK. Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase. J Cell Biol 135: 1609–1617, 1996.[Abstract/Free Full Text]
  29. Martinez-Calatrava MJ, Gonzalez-Sanchez JL, Zabena C, Martinez-Larrad MT, Luque-Otero M, Serrano-Rios M. Is the plasminogen activator inhibitor-1 gene a candidate gene predisposing to hypertension? Results from a population-based study in Spain. J Hypertens 25: 773–777, 2007.[Web of Science][Medline]
  30. Mehta PK, Griendling KK. Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol 292: C82–C97, 2007.[Abstract/Free Full Text]
  31. Moses S, Franzen A, Lovdahl C, Hultgardh-Nilsson A. Injury-induced osteopontin gene expression in rat arterial smooth muscle cells is dependent on mitogen-activated protein kinases ERK1/ERK2. Arch Biochem Biophys 396: 133–137, 2001.[CrossRef][Web of Science][Medline]
  32. Mulvany MJ. Is there a role for the vascular renin-angiotensin system in the determination of vascular structure? Blood Vessels 28: 224–230, 1991.[Web of Science][Medline]
  33. Nerurkar SS, Olzinski AR, Frazier KS, Mirabile RC, O'Brien SP, Jing J, Rajagopalan D, Yue TL, Willette RN. P38 MAPK inhibitors suppress biomarkers of hypertension end-organ damage, osteopontin and plasminogen activator inhibitor-1. Biomarkers 12: 87–112, 2007.[CrossRef][Web of Science][Medline]
  34. Oparil S, Zaman MA, Calhoun DA. Pathogenesis of hypertension. Ann Intern Med 139: 761–776, 2003.[Free Full Text]
  35. Panda D, Kundu GC, Lee BI, Peri A, Fohl D, Chackalaparampil I, Mukherjee BB, Li XD, Mukherjee DC, Seides S, Rosenberg J, Stark K, Mukherjee AB. Potential roles of osteopontin and alphaVbeta3 integrin in the development of coronary artery restenosis after angioplasty. Proc Natl Acad Sci USA 94: 9308–9313, 1997.[Abstract/Free Full Text]
  36. Park PJ, Cao YA, Lee SY, Kim JW, Chang MS, Hart R, Choi S. Current issues for DNA microarrays: platform comparison, double linear amplification, and universal RNA reference. J Biotechnol 112: 225–245, 2004.[CrossRef][Web of Science][Medline]
  37. Passegue E, Jochum W, Behrens A, Ricci R, Wagner EF. JunB can substitute for Jun in mouse development and cell proliferation. Nat Genet 30: 158–166, 2002.[CrossRef][Web of Science][Medline]
  38. Pulver RA, Rose-Curtis P, Roe MW, Wellman GC, Lounsbury KM. Store-operated Ca2+ entry activates the CREB transcription factor in vascular smooth muscle. Circ Res 94: 1351–1358, 2004.[Abstract/Free Full Text]
  39. Pulver-Kaste RA, Barlow CA, Bond J, Watson A, Penar PL, Tranmer B, Lounsbury KM. Ca2+ source-dependent transcription of CRE-containing genes in vascular smooth muscle. Am J Physiol Heart Circ Physiol 291: H97–H105, 2006.[Abstract/Free Full Text]
  40. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest 97: 1916–1923, 1996.[Web of Science][Medline]
  41. Rao GN, Katki KA, Madamanchi NR, Wu Y, Birrer MJ. JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells. J Biol Chem 274: 6003–6010, 1999.[Abstract/Free Full Text]
  42. Rapp JP. Genetic analysis of inherited hypertension in the rat. Physiol Rev 80: 135–172, 2000.[Abstract/Free Full Text]
  43. Rapp JP, Dene H. Development and characteristics of inbred strains of Dahl salt-sensitive and salt-resistant rats. Hypertension 7: 340–349, 1985.[Abstract/Free Full Text]
  44. Rapp JP, Wang SM, Dene H. A genetic polymorphism in the renin gene of Dahl rats cosegregates with blood pressure. Science 243: 542–544, 1989.[Abstract/Free Full Text]
  45. Rysa J, Leskinen H, Ilves M, Ruskoaho H. Distinct upregulation of extracellular matrix genes in transition from hypertrophy to hypertensive heart failure. Hypertension 45: 927–933, 2005.[Abstract/Free Full Text]
  46. Sawa H, Lundgren C, Sobel BE, Fujii S. Increased intramural expression of plasminogen activator inhibitor type 1 after balloon injury: a potential progenitor of restenosis. J Am Coll Cardiol 24: 1742–1748, 1994.[Abstract]
  47. Smeda JS, Payne GW. Alterations in autoregulatory and myogenic function in the cerebrovasculature of Dahl salt-sensitive rats. Stroke 34: 1484–1490, 2003.[Abstract/Free Full Text]
  48. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231–236, 1994.[CrossRef][Medline]
  49. Tank JE, Moe OW, Henrich WL. Abnormal regulation of proximal tubule renin mRNA in the Dahl/Rapp salt-sensitive rat. Kidney Int 54: 1608–1616, 1998.[CrossRef][Web of Science][Medline]
  50. Weissberg PL, Cary NR, Shanahan CM. Gene expression and vascular smooth muscle cell phenotype. Blood Press Suppl 2: 68–73, 1995.[Medline]
  51. Wellman GC, Cartin L, Eckman DM, Stevenson AS, Saundry CM, Lederer WJ, Nelson MT. Membrane depolarization, elevated Ca2+ entry, and gene expression in cerebral arteries of hypertensive rats. Am J Physiol Heart Circ Physiol 281: H2559–H2567, 2001.[Abstract/Free Full Text]
  52. Whitebread S, Mele M, Kamber B, de Gasparo M. Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem Biophys Res Commun 163: 284–291, 1989.[CrossRef][Web of Science][Medline]
  53. Whitmarsh AJ, Davis RJ. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 74: 589–607, 1996.[CrossRef][Web of Science][Medline]
  54. Wong C, Wellman TL, Lounsbury KM. VEGF and HIF-1alpha expression are increased in advanced stages of epithelial ovarian cancer. Gynecol Oncol 91: 513–517, 2003.[CrossRef][Web of Science][Medline]
  55. Workman C, Jensen LJ, Jarmer H, Berka R, Gautier L, Nielser HB, Saxild HH, Nielsen C, Brunak S, Knudsen S. A new non-linear normalization method for reducing variability in DNA microarray experiments. Genome Biol 3: research0048.1–research0048.16, 2002.
  56. Yamamoto M, Aoyagi M, Azuma H, Yamamoto K. Changes in osteopontin mRNA expression during phenotypic transition of rabbit arterial smooth muscle cells. Histochem Cell Biol 107: 279–287, 1997.[CrossRef][Web of Science][Medline]
  57. Zhou MS, Adam AG, Jaimes EA, Raij L. In salt-sensitive hypertension, increased superoxide production is linked to functional upregulation of angiotensin II. Hypertension 42: 945–951, 2003.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures
Right arrow All Versions of this Article:
294/2/H1075    most recent
00913.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rose, P.
Right arrow Articles by Lounsbury, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rose, P.
Right arrow Articles by Lounsbury, K. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.