gly96/IEX 1 is a growth- and apoptosis-regulating, immediate early gene that is widely expressed in epithelial and vascular tissues. In vascular tissues, expression of the gene is induced by mechanical stretch, and overexpression of the gene prevents injury-induced vascular smooth muscle hypertrophy and neointimal hyperplasia. We now show that deletion of the gly96/IEX-1 gene in mice is associated with development of elevated blood pressure, cardiac hypertrophy, and diminished fractional shortening of the left ventricle. Systolic blood pressure in conscious male gly96/IEX-1−/− mice is 20–25 mmHg higher than in gly96/IEX-1+/+ mice. Serum and/or urine concentrations of sodium, potassium, creatinine, angiotensin II, corticosterone, aldosterone, epinephrine, norepinephrine, prostaglandin E2, thromboxane B2, prostaglandin-6-keto-1α, nitrites and nitrates, cAMP, and cGMP are normal in gly96/IEX-1−/− mice. Alterations in dietary sodium intake do not alter blood pressure in gly96/IEX-1−/− mice. Aortic mRNAs for endothelial nitric oxide synthase, guanylate cyclase-α, and cGMP kinase-1 are increased in gly96/IEX-1−/− mice. Treatment with Nω-nitro-l-arginine methyl ester or l-arginine does not alter blood pressure in gly96/IEX-1−/− mice. Gly96/IEX-1−/− mice respond to infused sodium nitroprusside with decrements in blood pressure similar to those seen in wild-type littermate mice. In contrast to gly96/IEX-1 transgenic mice that have abnormalities in immune function, gly96/IEX-1−/− mice have normal lymphoid tissue architecture and a normal complement of T and B cells in lymphoid tissues. Ablation of the gly96/IEX-1 gene results in hypertension and cardiac hypertrophy, suggesting a novel role for this gene in cardiovascular physiology.
- elevated blood pressure
- lymphoid cells
the mouse gly96 gene is a growth factor- and serum-induced immediate early gene that encodes a 153-amino acid, glycosylated protein with a short half-life in serum-stimulated fibroblasts (6). The human ortholog of the mouse gly96 gene, IEX-1, is a radiation- and phorbol ester-induced immediate early gene that is expressed in a variety of epithelial and vascular tissues (8, 9, 18, 20, 26, 34). The expression of the IEX-1 gene is altered by factors such as serum, phorbol esters, ultraviolet and X-irradiation, pituitary adenylate cyclase-activating peptide, 1α,25-dihydroxyvitamin D3, nuclear factor-κB and p53, and cellular differentiation (15, 16, 18–20, 28, 31, 32). The promoter for the gly96/IEX-1 gene contains response elements for various transcription factors that modulate its activity (15, 16).
The biological role of this protein has been extensively examined in cell culture systems or after the overexpression of the gene in vivo (1, 11, 22, 34, 39–41). It is clear that IEX-1 plays a role in the control of growth and apoptosis in lymphoid cells, human embryonic kidney 293 cells, keratinocytes, and vascular smooth muscle cells (1, 2, 11, 20, 27, 33, 39, 41). In transgenic mice, in which IEX-1 is overexpressed specifically in T lymphocytes, impaired apoptosis in activated T cells, increased accumulation of effector/memory-like T cells, and susceptibility to a lupuslike autoimmune disease are observed (41).
Several lines of evidence suggest that the protein may play a role in vascular and cardiac tissues as well. First, the gly96/IEX-1 gene is prominently expressed in the vasculature (9, 18, 20). Second, the expression of the gene is rapidly increased on the application of mechanical stress to vascular smooth muscle cells maintained in culture, and in cardiac and aortic tissues on increasing blood pressure (8, 26, 34). In vivo studies have shown that the aortic banding in mice increases blood pressure and rapidly induces the messenger RNA for gly96/IEX-1 in vascular smooth muscle cells and cardiomyocytes within 6 h of the banding procedure (8). Third, forced expression of the gly96/IEX-1 gene blocks hypertrophy in vascular smooth muscle cells maintained in culture after the application of various stimuli, and overexpression of the gene is associated with protection from injury-associated vascular smooth muscle growth and neointimal hyperplasia (8, 34). We hypothesized that expression of the gly96/IEX-1 gene could play an important role in controlling cardiovascular function under normal circumstances and in various pathophysiological states and that ablation of the gene in the germ line could result in vascular abnormalities. No studies have previously been undertaken to examine the role of this gene in vascular and cardiac tissues using targeted gene ablation studies. Because gly96/IEX-1 could potentially be important in the control of cardiovascular function (8, 9, 20, 22, 24, 26, 34), we generated a mouse in which the gly96/IEX-1 gene was ablated in the germ line and examined it for alterations in cardiovascular physiology.
We now report that gly96/IEX-1 knockout mice exhibit an elevated blood pressure and cardiac hypertrophy. The cardiac ejection fraction and fractional shortening of the left ventricle are reduced in the knockout mice and the heart rate is increased. The elevated blood pressure appears to be independent of sodium intake and is not associated with changes in known vasoregulatory hormones and factors. Additionally, because others have reported altered T-cell function on overexpression of the gly96/IEX-1 gene, we examined lymphoid tissue architecture and T- and B-cell number and function in the gly96/IEX-1−/− mice and show that it is similar to that seen in wild-type littermate mice.
MATERIALS AND METHODS
Generation of a gly96/IEX-1 Null Mutant Mouse
Because the gly96/IEX-1 gene in the mouse is small (∼1.0 kb excluding the poly A+ tail, see Fig. 1A), we deleted the entire gene comprised of two exons and a small intervening intron (35). Exon 1 encodes the first 70 amino acids and exon 2 encodes the remaining 83 amino acids. A small intervening sequence, or intron, of 109 nucleotides separates the two exons. This approach ensured the lack of expression of RNA encoding the entire protein.
The methods used to generate a knockout mouse were as follows.
Generation of gly96/IEX-1 DNA lacking exons 1 and 2 and the intervening intron.
The sequence of the gly96/IEX-1 mouse was obtained from the Celera mouse database. Genomic sequence 5′ of exon 1 was amplified to generate a ∼4.0 kb pair DNA sequence using mouse strain 129 genomic DNA, PCR methods, and primers with Hpa1 and Xho1 sites at the 5′ and 3′ ends, respectively (see Fig. 1A; 3). The DNA product was treated with Hpa1 and Xho1 restriction endonucleases using conditions described by the manufacturer. The DNA obtained in this manner was purified by gel electrophoresis. This fragment is referred to as fragment A. Genomic sequence 3′ of exon 2 of the gly96/IEX-1 gene was amplified using mouse strain 129 genomic DNA and PCR with primers containing Not1 restriction endonuclease sites (see Fig. 1A). An ∼2.0 kb pair DNA product was treated with Not1 restriction endonuclease and was purified by gel electrophoresis. This fragment is referred to as fragment B.
Construction of the targeting vector.
Fragment A (see above) was cloned into site A of Hpa1 and Xho1 endonuclease-treated pKO Scrambler NTKV 1901 plasmid vector (Stratagene, La Jolla, CA; subsequently referred to as SiteA4kb-pNTKV). Fragment B was cloned into Not 1 cut SiteA4kbpNTKV to yield SiteA4kbpsiteB2kbp-pNTKV—the knockout vector. This is comprised of the mutant gly96/IEX-1 gene lacking exons 1 and 2 and intron 1 with a neomycin cassette in place of the two exons and one intron (see Fig. 1A).
Transfection of embryonic stem cells and Southern analysis of DNA.
The construct was linearized (concentration ∼40 μg/ml) and used to electroporate embryonic stem cells (ES cells) grown on mouse embryonic feeder cells (∼20–30 × 106 ES cells in 750 μl phosphate-buffered saline) (35). A 0.4-cm gap size cuvette, and a BioRad Gene Pulser (BioRad, Hercules, CA) at a setting of 230 V/500 μF (time constant 6–8 ms) were used for electroporation of ES cells with the knockout vector (33). The electroporated ES cells were grown on irradiated fibroblasts and subjected to antibiotic selection with G418 (350 μg/ml) and 0.2 μM 1–2-deoxy[2-fluoro-β-d-arabinofuranosyl] 5-iodouracil. Antibiotic-resistant clones were identified and analyzed by Southern blotting using a radiolabeled probe (Fig. 1A). Two clones out of 384 ES clones were found by Southern analysis to contain the mutant construct at the appropriate site (see Fig. 1B).
Embryonic stem cells containing the mutant construct were expanded and injected into blastocysts derived from C57BL/6 mice, which were transferred to the uterus of BCBA female mice. Chimeric black/agouti mice and subsequently heterozygous knockout (gly96/IEX-1 +/−) agouti mice were obtained by appropriate breeding methods.
Southern Blotting, PCR Analysis of Normal and Mutant Alleles, and RT-PCR
Genomic DNA was isolated from tail samples of mice using a kit from Qiagen (Valencia, CA). Southern blotting was carried out as described using an oligonucleotide probe that hybridizes to the gly96/IEX-1 gene at the position indicated in Fig. 1A (3). Tissue total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA). PCR and RT-PCR were carried out using the following primers, and PCR and RT-PCR kits from Roche Applied Sciences (Indianapolis, IN) or Clontech BD Biosciences (Palo Alto, CA).
DNA primers for PCR of genomic Gly96 DNA.
Primers were as follows: 5′-GCA GTT TTG TGT CCG TGT GCT C-3′ (gly96 forward primer); 5′-TTC TTC GGA CTG TGA CCC ATC G-3′ (gly96 reverse primer); 5′-GGG CTG ACC GCT TCC TCG TGC TTT-3′ (Neo cassette reverse primer).
Conditions for PCR were 95°C for 5 min (1 cycle); 95°C for 30 s, 58°C for 1 min, 72°C for 2 min (35 cycles); 72°C for 5 min (1 cycle). The expected DNA PCR products were 1,159 bp the normal wild-type allele and 926 bp for the mutant allele.
PCR primers for RT-PCR.
Size of PCR product = 300 bp. 5′-TGT TCG CCA TCA TCT TCT GGC-3′ (forward primer); 5′-TTC TTC GGA CTG TGA CCC ATC G-3′ (reverse primer).
ENDOTHELIAL NITRIC OXIDE SYNTHASE.
Size of PCR product = 512 bp. 5′-ATG CTC CCA ACT GGA CCA TCT C-3′ (forward primer); 5′-AAG TGA CAC AAT CCC TCT TTC CG-3′ (reverse primer).
CYCLIC GUANOSINE MONOPHOSPHATE KINASE 1.
Size of PCR product = 411 bp. 5′-CGA AGA GAC CCA CTA TGA AAA TGG-3′ (forward primer); 5′-ACT CGT CCG AAA CCT CCA ACT C-3′ (reverse primer).
GUANYLATE CYCLASE α.
Size of PCR product = 798 bp. 5′-ATG CTC AAC GCT CTC TAC ACT CG-3′ (forward primer); 5′-CAC AAC TAT GCC CCC AAA AGT C-3′ (reverse primer).
Size of PCR product = 500 bp. 5′-GTG GGC CGC TCT AGG CAC CAA-3′ (forward primer); 5′-CTC TTT GAT GTC ACG CAC GAT TTC-3′ (reverse primer).
Diets and blood pressure measurement.
The Institutional Animal Care and Use Committee of the Mayo Clinic approved all animal protocols. Five- to six-week-old age-matched male gly96/IEX-1+/+ and gly96/IEX-1−/− mice (n = 6/group) were fed a normal sodium diet (0.3% Na diet, diet number 2016, Harlan-Teklad, Madison, WI) and systolic blood pressures were measured daily in conscious mice, using a four-mouse automated tail cuff instrument with computerized data acquisition for a period of 14 wk (Visitech System, Apex, NC). After a 2-wk acclimation period, all mice were fed a normal sodium diet for 2 wk, and tail cuff blood pressures were measured and recorded daily. The mice were then placed in metabolic balance cages, and urine samples were collected on the last 2 days (days 13–14) of normal sodium diet. The mice were then fed a high-sodium diet (3% Na, diet number 92012, Harlan-Teklad) for 2 wk. Blood pressures were measured daily, and urine samples were collected on the last 2 days of high-sodium intake. All animals were then fed a normal sodium diet (0.3% Na) for 2 wk. The nonselective nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 2 mg/ml) was added to the drinking water. Blood pressures were measured daily for 2 wk, and urine samples were collected on the last 2 days of the l-NAME treatment period. These mice were then fed a normal sodium diet for 2 wk and blood pressures were again recorded daily. Subsequently, these mice were fed a low-sodium diet (0.03% Na, diet number 90228, Harlan-Teklad) for 2 wk. Tail cuff blood pressures were recorded daily, and 24-h urine samples were collected the last 2 days of the low-sodium diet. The mice were then fed a normal sodium diet for 2 wk. At the end of this period, the mice were anesthetized with Inactin (130 mg/kg), a blood sample was taken, and the heart was removed and weighed.
In another group of mice, the effect of l-arginine administration on blood pressure was studied. Male gly96/IEX-1+/+ (n = 6) and gly96/IEX-1−/− (n = 6) mice (age ∼12 wk) were fed a normal sodium diet and, after a 2-wk acclimation period, l-arginine (2%) was added to the drinking water. Blood pressures were measured daily by tail cuff for 2 wk, and urine samples were collected the last 2 days of l-arginine treatment.
Measurement of blood pressure by telemetry.
Twelve-week-old male gly96/IEX-1+/+ (n = 3) and gly96/IEX-1−/− mice (n = 5) were anesthetized with 2% isoflurane and catheters linked to a telemetry system (Data Sciences International, St. Paul, MN) were inserted in the left carotid artery. All animals were allowed to recover from surgery for 3 days and then blood pressures were measured for 10 s every 5 min. These blood pressures were averaged every 12 h (6 PM–6 AM, 6 AM–6 PM) for 5 days.
Echocardiography in mice.
Eighteen gly96/IEX-1−/− and 17 gly96/IEX-1+/+ mice (age ∼16 wk) underwent echocardiography after being anesthetized with avertin as described previously (29). Two-dimensional targeted M-mode echocardiography was performed (GE Vingmed System, Horten, Norway) with a 10-MHz probe. Left ventricular end-diastolic and end-systolic dimensions as well as interventricular septum dimensions (IVSd, IVSs) and posterior wall dimensions (PWd, PWs) both in systole and diastole were measured. Fractional wall shortening, ejection fraction, and LV mass were calculated as described previously.
Infusion of sodium nitroprusside.
Acute studies were performed in anesthetized gly96/IEX-1−/− and gly96/IEX-1+/+ (n = 11/group) mice (age ∼15–18 wk) to determine the blood pressure response to sodium nitroprusside. Mice were anesthetized with Inactin (130 mg/kg), and a catheter was placed in the jugular vein for infusion and the carotid artery for measuring blood pressure. The mice received an infusion of 0.9% NaCl at a rate of 0.5 ml/h throughout the experiment. After a 45-min stabilization period, progressively increasing doses (1, 3, 10, 30, 100 μg/kg) of sodium nitroprusside were administered as intravenous boluses (0.2 ml) at 4-min intervals. The maximal decreases in blood pressure were compared between gly96/IEX-1−/− and gly96/IEX-1+/+ mice.
Chemical analysis of urine and blood.
Sodium concentrations in serum and urine were measured by flame photometry (Instrumentation Laboratory, Lexington, MA). Creatinine concentrations in serum and urine were measured with a creatinine analyzer (Beckman Instruments, Fullerton, CA). Nitrate/nitrite concentrations in urine and plasma were determined using a kit from Cayman Chemicals (Ann Arbor, MI). Plasma angiotensin II and urinary corticosterone, aldosterone, PGF2, TBX, and 6-keto-PGF1α in urine were determined using kits from Cayman Chemicals (Ann Arbor, MI). Urinary cAMP and cGMP were measured using kits from Biomedical Technologies (Stoughton, MA). Epinephrine and norepinephrine were measured in urine using kits from Alpco Diagnostics (Windham, NH).
Histological evaluation of the heart and kidneys.
Male gly96/IEX-1+/+ (n = 2) and gly96/IEX-1−/− mice (n = 2) were anesthetized with pentobarbital sodium, and their hearts and kidneys were removed, fixed, and embedded in paraffin. The paraffin samples were cut into 6-μm sections and stained with hematoxylin and eosin. Cardiomyocyte diameter was measured in 10 transverse sections/group. Renal histology was assessed.
Flow cytometric analysis of lymphoid tissues.
Spleens, thymi, inguinal subcutaneous lymph nodes, mesenteric lymph nodes, and femurs were dissected from gly96/IEX-1+/+ and gly96/IEX-1−/− littermate mice aged 8–12 wk. Spleens, thymi, and lymph nodes were weighed and then mechanically dissociated in 1 ml of FACs buffer (PBS, pH 7.4; 0.2% BSA, 0.02% NsN2). Bone marrow was flushed from femurs using a 1-ml syringe containing FACs buffer. The resulting cell suspensions were filtered through a 45 μM nylon mesh, and cells were counted with a hematocytometer. For spleen, thymus, and bone marrow, erythrocytes were removed by 5 min incubation in ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA) followed by washing in FACs buffer. All cell suspensions were adjusted to 2 × 106 cells/ml and 100-μl aliquots were incubated with a panel of FITC, phycoerythrin (PE), or biotin-coupled monoclonal antibodies for 30 min at 4°C. Where appropriate, biotinylated antibodies were revealed by secondary incubation with streptavidin-PerCP for 20 min at 4°C. Antibody-labeled samples were analyzed immediately by two- or three-color flow cytometry using a FACscan flow cytometer and Cellquest software (BD Pharmingen, San Diego, CA). Viability was determined by flow cytometry using propidium iodide staining. For each cellular subset, the proportionate representation in each sample was calculated for each of the lymphoid organs and mean ± SD results for gly96/IEX-1+/+ and gly96/IEX-1−/− mice were compared using two-tailed, unpaired Student’s t-test. The monoclonal antibodies used for flow cytometric analysis (all purchased from BD Pharmingen) were anti-mouse CD4-FITC (Clone RM4–4), anti-mouse CD8-PE (clone 53–6.7), anti-mouse CD45RB-biotin (clone 1A6), anti-mouse CD25-PE (clone PC-61), anti-mouse T-cell receptor β-biotin (clone H57–597), anti-mouse CD19-PE (clone 1D3), anti-mouse IgD-FITC (clone 11–26c-2a), anti-I-Ab-biotin (clone AF6–120.1), anti-mouse CD11b-PE (clone M1/70), and anti-mouse CD11c-biotin (clone HL3).
Activation-induced apoptosis of lymphocyte populations was examined in vitro by culturing erythrocyte-depleted splenocytes in round-bottom tissue culture plates (2.5 × 105 cells/well) for 72 h in the presence of 1) no stimulation, 2) T-cell-specific stimulation, hamster anti-mouse CD3ε (2C11, BD Pharmingen) at low (0.01 μg/ml) and high (1.0 μg/ml) concentration, 3) B cell stimuli, LPS 10 ng/ml alone or in combination with hamster anti-mouse CD40 (HM40–3, 1.0 μg/ml; BD Pharmingen). Cell suspensions were stained for viability, early apoptosis, and late apoptosis/necrosis using the Vybrant Apoptosis Assay kit no. 10 (Molecular Probes, Eugene, OR) and analyzed by three-color flow cytometry according to manufacturer’s instructions.
Immunohistochemical analysis of lymphoid organs.
Freshly dissected spleens, thymi, inguinal subcutaneous lymph nodes, and mesenteric lymph nodes were placed in embedding medium (OCT compound) and frozen in liquid nitrogen-cooled isopentene. Six-micrometer cryosections were prepared on silane-coated slides were fixed in acetone (−20°C for 10 min), air-dried, and rinsed in PBS. Endogenous peroxidase activity was blocked with 0.3% H2O2 in PBS for 10 min and slides were rinsed in PBS. Normal goat serum (5% in PBS) was applied for 1 h followed by blockade of endogenous biotin (Vector Laboratories, Burlingame, CA) and incubation with a panel of biotinylated monoclonal antibodies for 1 h at room temperature. Slides were rinsed in PBS, incubated with ABC complex (Vector Laboratories) for 45 min, rinsed again, and DAB (brown) substrate solution was applied and allowed to develop to the desired color intensity. The monoclonal antibodies, isotype controls, and other detection reagents used for immunohistochemistry were anti-mouse CD4-biotin (clone H129.19), anti-mouse CD8α-biotin (clone 53–6.7), anti-mouse CD11c-biotin (clone HL3), anti-mouse panendothelial cell antigen-biotin (clone MECA-32), anti-mouse CD45R/B220-biotin (clone RA3–6B2), hamster IgG1 κ-biotin (clone A19–3), rat IgG2a, k-biotin (clone R35–95; all purchased from BD Pharmingen), and biotinylated peanut agglutinin (Vector Laboratories).
Statistical comparisons between groups were made using unpaired or paired t-tests. A P value of <0.05 was considered statistically significant.
Characterization of the Genotype of gly96/IEX-1−/− Mice and Assessment of mRNA Expression in Tissues
To determine the function of the gene and its product in vivo in the intact animal, we ablated the mouse gly96/IEX-1 gene in mouse embryonic stem cells using homologous recombination methods (Fig. 1A; 35). Knockout mice (gly96/IEX-1−/−) showed the appropriate mutant alleles on Southern analysis (Fig. 1B), polymerase chain reaction analysis of DNA (Fig. 1C), and absent gly96/IEX-1 tissue mRNA by reverse transcription and PCR analysis (Fig. 1D). Of note, no RNA of the gly96/IEX-1 gene was noted in gly96/IEX-1−/− mice.
Cardiovascular Phenotype of gly96/IEX-1−/− and gly96/IEX-1+/+ Mice
Gly96/IEX-1−/− mice were morphologically normal on inspection, reproduced normally, and grew at a normal rate. Because gly96/IEX-1 is expressed in endothelial cells and vascular smooth muscle cells (9, 34), we measured arterial blood pressure in these mice.
Gly96/IEX-1−/− mice (age 8 wk) had elevations of systemic arterial pressure as measured by tail cuff plethysmography (systolic blood pressure, 154.7 ± 3.6 mmHg, n = 6, mean ± SE, in gly96/IEX-1−/− male mice vs. 124 ± 5.1 mmHg in gly96/IEX-1+/+ mice, n = 6, P < 0.0008). Blood pressures were also measured continuously in conscious gly96/IEX-1+/+ (n = 3) and gly96/IEX-1−/− (n = 5) mice using a telemetry system. As shown in Fig. 2, mean arterial blood pressure, when measured continuously, is significantly higher by 20–25 mmHg in the gly96/IEX-1−/− mice than in the gly96/IEX-1+/+ mice. The heart rate of knockout mice was higher than that of wild-type mice (Table 1). Fractional shortening and left ventricular ejection fraction were reduced in gly96/IEX-1−/− mice showing possible heart failure (Table 1).
To evaluate the presence and extent of cardiac hypertrophy, we measured heart weight and performed echocardiography. At 16 wk of age there was an increase in heart weight (4.80 ± 0.22 mg/g body wt in gly96/IEX-1−/− mice vs. 3.97 ± 0.16 mg/g body wt in gly96/IEX-1+/+ mice, P < 0.01). An increase in left ventricular (LV) diameter in diastole and an increase in LV mass (Table 1) demonstrates the presence of LV hypertrophy.
The presence of cardiac hypertrophy on echocardiography was confirmed by the measurement of cardiomyocyte diameter on microscopy. Histological examination and measurement of cardiomyocyte diameter demonstrated that the myofibril diameter was significantly greater (103.5 ± 4.0 μm) in the gly96/IEX-1−/− mice than in the gly96/IEX-1+/+ mice (67.2 ± 1.9 μm, P < 0.0001).
Effect of Variations in Dietary Sodium on Blood Pressure
We next assessed whether factors known to contribute to hypertension were operative in these mice. Because some rodents develop hypertension on ingesting salt (23), we determined whether blood pressure changed on varying dietary salt intake in gly96/IEX-1−/− mice. Gly96/IEX-1−/− mice fed a normal salt diet had a systolic blood pressure of 154.7 ± 3.6 mmHg and, after ingestion of a high-salt diet, had a systolic blood pressure of 152.7 ± 3.0 mmHg, P = not significant (NS). Gly96/IEX-1−/− mice fed a normal-salt diet had a systolic blood pressure of 143.8 ± 1.5 mmHg and, after ingestion, of a low-salt diet had a systolic blood pressure 144.0 ± 3.7 mmHg, P = NS. Wild-type mice did not show changes in blood pressure on dietary salt manipulation. Thus arterial blood pressure in gly96/IEX-1−/− mice is independent of dietary salt intake.
Concentrations of Blood Pressure Regulating Factors in gly96/IEX-1−/− Mice
A variety of hormones such as catecholamines, glucocorticoids, and mineralocorticoids are known to influence arterial blood pressure. In addition, factors produced locally in the blood vessel wall alter arterial tone. To determine whether such factors/hormones are altered in gly96/IEX-1−/− mice, we measured urine or serum concentrations of appropriate hormones or their metabolites in gly96/IEX-1−/− mice and wild-type littermates (Table 2). Gly96/IEX-1−/− mice had normal renal function. Serum and urine sodium and potassium concentrations and excretion were similar in knockout and global wild-type mice, suggesting normal adrenal cortical physiology in knockout mice. This is supported by similar plasma angiotensin II concentrations and normal corticosterone and aldosterone excretion in the urine of gly96/IEX-1−/− mice when compared with gly96/IEX-1+/+ mice. Urinary catecholamine excretion was similar in gly96/IEX-1−/− and gly96/IEX-1+/+ mice. Body weights were similar in the gly96/IEX-1−/− and gly96/IEX-1+/+ mice. Urinary PGE2, prostaglandin 6-keto-1α, and thromboxane B2 excretion were normal in knockout and wild-type mice. Urinary nitrites and nitrates, cAMP, and cGMP were similar in the urine of gly96/IEX-1−/− and wild-type mice.
Endothelial Nitric Oxide Synthase System in the Aorta of gly96/IEX-1−/− Mice
Endothelial nitric oxide release is important in the control of vascular tone. To assess whether any abnormalities were present in the nitric oxide signaling pathway in blood vessels, we measured endothelial nitric oxide synthase mRNA, and the mRNA for molecules whose synthesis is altered by nitric oxide in the blood vessel wall. There was an increase in endothelial nitric oxide synthase, guanylate cyclase, and cGMP kinase-1 mRNA concentrations in gly96/IEX-1−/− mouse aortic tissue when compared with that observed in wild-type littermate mice (Fig. 3, A–D).
The presence of upregulated endothelial nitric oxide synthesis and an increase in mRNA concentrations of molecules induced by nitric oxide suggest the presence of a compensatory response to the elevated blood pressure or the presence of a resistance to endogenous nitric oxide. To further assess this, we infused increasing amounts of sodium nitroprusside intravenously into knockout and wild-type mice and measured changes in blood pressure in response to the administered nitric oxide donor. As shown in Table 3, there were identical decrements in mean arterial pressure in response to given amounts of sodium nitroprusside. This suggests that the blood vessel is able to respond to nitric oxide in a normal fashion.
To further assess the presence of abnormal nitric oxide signaling in the blood vessel wall of gly96/IEX-1 knockout mice, we administered knockout or wild-type mice l-NAME or l-arginine over a period of 2 wk. In normal wild-type mice there was a small (but statistically insignificant) increase in blood pressure after the administration of l-NAME. No change in blood pressure was observed in the knockout mice. l-arginine feeding did not change blood pressure in either knockout or wild-type mice.
Histology of the Aorta and Medium and Small Blood Vessels in the Kidney
Aortic tissue form gly96/IEX-1−/− and gly96/IEX-1+/+ mice appeared histologically identical (data not shown). Renal tissue obtained from the gly96/IEX-1 knockout animals showed no significant interstitial fibrosis, tubular atrophy, or interstitial inflammation. The glomeruli appeared normal. There was no significant medial hypertrophy or sclerosis of interstitial arteries (data not shown). The mean media to vessel wall diameter was 0.27 in gly96/IEX-1−/− animals and 0.22 gly96/IEX-1+/+ mice (P = NS). Arterioles did not contain any significant hyaline deposits. On the basis of these considerations, there was no evidence of hypertensive nephrosclerosis in gly96/IEX-1−/− mice.
Immunological Assessment of gly96/IEX-1−/− Mice
Transgenic mice overexpressing IEX-1 in lymphoid cells have altered immune function (41). To determine the role of gly96/IEX-1 in normal development and homeostasis of major compartments of the cognate immune system, the composition and architecture of the spleen, subcutaneous and mesenteric lymph nodes, thymus and bone marrow were examined in groups of gly96/IEX-1+/+ and gly96/IEX-1−/− mice. There were no differences in the anatomy and weight of spleens, lymph nodes, and thymi from wild-type and knockout mice ranging in age between 8 and 12 wk (data not shown). No significant differences were observed between lymphoid tissues from gly96/IEX-1+/+ and gly96/IEX-1−/− animals with regard to overall cell viability and proportions of CD4 T cells/thymocytes; CD8 T cells/thymocytes; double negative and double positive thymocytes; regulatory (CD25pos) CD4 T cells; naive (CD45RBhi) CD4 and CD8 T cells; B cells; naive (IgDhi) B cells; “myeloid” (CD8neg) and “lymphoid” (CD8pos) dendritic cells, and monocyte/macrophage (Table 4). The architecture of spleens, subcutaneous and mesenteric lymph nodes, and thymi from adult gly96/IEX-1−/− mice was compared with that of wild-type littermates by immunostaining of frozen tissue sections for proteins distinguishing T cell subsets (CD4 and CD8), B cells and germinal center B cells [B220 and peanut agglutinin (PNA)], dendritic cells (CD11c), endothelial cells (panendothelial antigen). In organs examined by immunohistochemistry, the distribution of positively stained cells for T cell, B cell, dendritic cell, and endothelial markers was comparable between gly96/IEX-1+/+ and gly96/IEX-1−/− specimens. Representative examples for the spleen are provided in Fig. 4, A–L). In vitro stimulation of T and B cells derived from spleens of gly96/IEX-1+/+ and gly96/IEX-1−/− animals was carried out to examine rates of activation-induced apoptosis of lymphocytes in the presence and absence of gly96/IEX-1. In these experiments, the proportions of viable, early apoptotic, and late-apoptotic/necrotic cells were closely comparable in unstimulated 72 h splenocyte cultures from the two groups of mice and after high-level T-cell and both high- and low-level B-cell stimulation. Low-level stimulation of T cells was variably associated with a modest relative increase in early apoptotic cells in gly96/IEX-1−/−splenocyte cultures (data not shown).
The gly96/IEX-1 gene is widely expressed in epithelial and endothelial tissues (9, 20, 22) and expression is particularly prominent in the vasculature (9, 18, 20). The amount of gly96/IEX-1 mRNA is rapidly increased in vascular smooth muscle cells on the induction of mechanical stress and on the increase in blood pressure (8, 26, 34). Furthermore, it appears that the gly96/IEX-1 gene blocks hypertrophy in vascular smooth muscle cells after the application of various stimuli, and overexpression of the gene is associated with protection from injury-associated vascular smooth muscle growth and neointimal hyperplasia (8, 34). These observations would suggest that expression of the gly96/IEX-1 gene might play an important role in the maintenance of normal cardiovascular function under normal circumstances or in pathophysiological circumstances. Before the current observations, there have been no reports concerning the effect of ablation of the gly96/IEX-1 gene on the cardiovascular function and anatomy in vivo.
We show that ablation of the gly96/IEX-1 gene in mice results in elevated blood pressure and cardiac hypertrophy. The mechanisms by which gly96/IEX-1 regulates vascular tone are unknown. Because blood pressure is determined by cardiac output and by peripheral vascular resistance, either one of these could be responsible for the pathogenesis of hypertension in gly96/IEX-1 knockout mice. Increased cardiac output cannot account for the elevation of blood pressure, because ejection fraction is slightly, but significantly, decreased on echocardiography. Hence, it is likely that changes in peripheral resistance are the cause of the increase in blood pressure.
We assessed whether there were changes in the concentrations of known blood pressure-regulating hormones such as catecholamines, angiotensin II, aldosterone, and glucocorticoids in knockout and wild-type mice. None of the factors that increase vascular tone, such as catecholamines or angiotensin II were increased in knockout mice compared with wild-type mice. There were no changes in the urinary excretion of substances associated with an increase sodium retention and in blood volume such as glucocorticoids or aldosterone. The renin-angiotensin system appeared similar in knockout and wild-type mice based on the following observations: similar concentrations of serum angiotensin II; similar urinary aldosterone excretion; normal urinary excretion and serum concentrations of sodium and potassium; and a normal juxtaglomerular apparatus on renal histology.
The excretion of vasoregulatory prostaglandins was normal in gly96/IEX-1−/− mice compared with wild-type littermate mice. Neither the urinary excretion of vasodilator prostaglandins such as PGE2 or PGF 6-keto-1α were different in gly96/IEX-1−/− and gly96/IEX-1+/+ mice, nor were concentrations of the vasoconstrictor thromboxane-B2 different among the two groups.
The increase in expression of genes involved in nitric oxide function in the aorta is of interest. Although not tested, presumably similar increases in the endothelial nitric oxide signaling are present in smaller arteries that are responsible and might play a major role in the control of blood pressure. Expression of the mRNAs of endothelial nitric oxide synthase, guanylate cyclase-α, and cGMP kinase-1 is increased in gly96/IEX-1−/− mice either as a compensatory response to the hypertension or as a result of modulation of the nitric oxide pathway by gly96/IEX-1. Increased aortic tissue nitric oxide signaling, and presumably increased nitric oxide signaling in smaller blood vessels, fails to reduce the elevated blood pressure. Of note, similar increases in endothelial nitric oxide synthase expression are seen in Dahl salt-sensitive rats (36). The administration of pharmacological amounts of a nitric oxide donor, sodium nitroprusside, however, elicits a similar hypotensive response in knockout and control mice. It is most likely that the changes observed in the gly96/IEX-1−/− mice are present as a result of a compensatory response to the increase in blood pressure, but it is possible that a lack of response to endogenous nitric oxide signaling pathways could be playing a role in blood pressure elevations seen in this mouse. It is interesting to note that IEX-1 interacts with calcium-modulating cyclophilin ligand (CAML), a protein known to alter intracellular calcium concentrations (4, 14, 21, 37). Additionally, recent information suggests that after stimulation by angiotensin II, the activity of the angiotensin receptor 1 with respect to the activation of the nuclear factor of activated T cells (NF-AT), is modulated by CAML in cells (12). It is possible that through its interactions with CAML, IEX-1 may alter intracellular calcium and vascular smooth muscle cell tone and cardiomyocyte growth.
The myocardial hypertrophy present in gly96/IEX-1−/− mice is most likely due to persistently elevated blood pressure in the knockout mice. Altered myocardial remodeling from changes in the rate of apoptosis as a result of absent gly96/IEX-1 expression might play a role as well (1, 2, 8, 33, 34, 38). The decrease in left ventricular contractility is of interest, but the precise mechanism for this phenomenon remains to be elucidated. The altered myocardial contractility observed on echocardiography could be secondary to heart failure induced by elevated blood pressure or to an intrinsic effect of gly96/IEX-1 on cardiac contractility. Gly96/IEX-1 overexpression in vascular smooth muscle cells blocks hypertrophy seen after treatment with phenylephrine and after the induction of injury (8, 34). In mice in which the gly96/IEX-1 gene is ablated, there are no changes in the histology of blood vessels. Whether there are differences in the patterns of response to injury remains to be determined.
Numerous studies, performed in cells maintained in culture or in transgenic mice in which the gly96/IEX-1 is expressed in high concentrations in lymphoid cells have shown that it regulates growth and apoptosis in a variety of cell types (1, 10, 11, 20, 22, 30, 33, 34, 38–41). Our results demonstrate that gly96/IEX-1 is not essential for normal development of the major lymphoid organs. During health, gly96/IEX-1 has no nonredundant functions in maintaining normal proportions of thymocyte and T-cell subsets, in determining the balance between naive and memory lymphocytes, or in regulating the generation and distribution of antigen-presenting cell populations. In addition, our in vitro experiments have not demonstrated striking alterations in activation-induced apoptosis of T and B lymphocytes. These observations do not rule out an in vivo role for gly96/IEX-1 in regulating immune responses to infection, neoplasia, vaccination, self antigens, or transplanted tissues, the elucidation of which will be greatly facilitated by further studies in gly96/IEX-1−/− animals.
Because hypertension affects 50 million individuals in the United States and 1 billion people worldwide and is the major cause of premature stroke, heart failure, and cardiovascular disease (5, 7), new information regarding the control of vascular tone is important. The contribution of environmental factors to blood pressure regulation is modest, and most hypertension has a genetic basis. The genetic contribution to the regulation of blood pressure varies from 25% in pedigree studies to 65% in twin studies, with about 7% of the total variance of diastolic blood pressure being due to environmental factors (13). In most affected subjects, the precise cause of hypertension is not known (essential hypertension) and essential hypertension is a polygenic disorder (17). In most cases of genetic hypertension, a single mutation in a gene is sufficient to produce the disease trait (13). An example of such a genetic condition is the syndrome of glucocorticoid-remediable aldosteronism (25) in which transmission of the disorder occurs in an autosomal dominant manner. Other genes have been linked to the regulation of blood pressure in humans, but their role in the pathogenesis of hypertension in the human population is uncertain. Identification of specific genes that influence blood pressure could be important with respect to the identification of those at risk for hypertension and to the development of drug therapy. Whether gly96/IEX-1 is such a gene, remains to be determined, but our studies suggest that it should be given consideration.
In summary, ablation of the gly96 gene in mice results in elevated blood pressure and cardiac hypertrophy and these results suggest that factors such as immediate early genes could be important in cardiovascular physiology and disease.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-25409 to R. Kumar.
We thank R. Bolterman, M. Manriquez, D. Meyers, and G. Harders for technical assistance.
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.
- Copyright © 2006 the American Physiological Society