Hypertension is a common disease and has complicated physiological mechanisms. Recently, evidence has shown that the immune system plays an important role.1,2 The autoantibodies against angiotensin AT1 receptor (AT1-RAb) were discovered by Fu et al3 in the sera of malignant patients and by Wallukat et al4 in preeclamptic and Liao et al5 in refractory hypertensive patients. The autoantibodies against AT1 receptor presented an agonist effect similar to angiotensin II (Ang II) in sera of patients with malignant and refractory hypertension.6–8 Later, Wang et al9 confirmed that the arterial structure changed in rats immunized by AT1 receptor peptide, which indicated the antibodies against AT1 receptor could play a role in the pathogenesis of hypertension. Ang II stimulates a rapid increase in the mRNA levels of c-fos to proliferate in vascular smooth muscle cells (VSMCs) via AT1 receptor binding,10 for which the Janus kinase signal transductors and activation of transcription (JAK-STAT) and nuclear factor-κB (NF-κB) were essential.11,12 In addition, the antibodies against AT1 receptor from immunized rats could induce proliferation of VSMCs.9 However, the underlying mechanism and whether the autoantibodies against AT1 receptor have an effect on proliferation of VSMCs remain unknown.
In the present study, we tested the possibility that the autoantibodies against AT1 receptor could account for the proliferation in VSMCs and studied further the signalling molecules.
This study was approved by the Ethics Committee of Union Hospital. All patients gave their oral informed consent and all tested animals were obtained legally. The patients with hypertension were recruited from the Institute of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. The 40 samples from hypertensive patients, without any drug therapy, were collected as described before.5
All solutions and reagents used in cell culture and activation procedure were endotoxin free. Anti-JAK2, anti-STAT1, anti-STAT3, antiphosphorylated JAK2 (pJAK2), antiphosphorylated STAT1 (pSTAT1), antiphosphorylated STAT3 (pSTAT3) and anti-NF-κB (P50) were purchased from Santa Cruz Biotechnology, USA. Medium and calf serum were purchased from Gibico, USA. Cell Proliferation Assay Kit was purchased from Roche and ECL kit was from Pierce, USA. Nitrocellulose membrane and nylon membrane were purchased from Whatman, UK. All other reagents were purchased from Sigma Chemical Company, USA.
Antigen peptide synthesis
The peptide corresponding to the sequence of the second extracellular loop of the human AT1 receptor positions 165aa-191aa (I-H-R-N-V-F-F-I-E-N-T-N-I-T-V-C-A-F-H-Y-E-S-Q-N-S-T-L) was synthesized by an automated, multiple solid phase, peptide synthesizer (PSSM-8 type; Shimadzu, Japan). The peptide was evaluated by high-performance liquid chromatography (HPLC) analysis on a Vydac C-18 column and 95% purity was achieved.
Measurement and preparation of the autoantibodies
We collected the sera from patients with primary hypertension (blood pressure ≥149/90 mmHg, obtained on at least two measurements, patients with secondary hypertension were excluded). The AT1-RAb titre was detected by ELISA method.5 The titre was shown by the value of P/N when the sera were diluted 1:40 ((P/N = (the A value of test - the A value of blank) / (the A value of negative control - the A value of blank)). Preparation of the IgG fraction was described elsewhere.13 The IgG fraction was loaded onto chromatographic column containing Sepharose 4B CNBr activated gel (Pharmacia, USA) to which the peptide corresponding to the second extracellular loop of the human AT1 receptor was covalently linked. The antibodies were eluted with 3 mol/L potassium thiocyanate (pH 7.4) followed by immediate superfiltration by Millipore Centriplus 50 000 r/min, changed and concentrated in phosphate buffered saline; the purity was assessed by SDS-PAGE.
VSMCs culture and identification
Adult Wistar rat aortic VSMCs were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% (v/v) new born serum, 10 mg/ml streptomycin and 100 U/ml penicillin at 37°C in a 5% CO2 enriched, humidified atmosphere.10 Cells from passages 5 and 6 were routinely subcultured 1:2 or 1:3 at 3-day intervals and the medium was changed at 2–3 day intervals. We identified the VSMCs by inverted phase contrast microscope and immunohistochemistry. VSMCs were fixed by propanone. Immunostaining was carried out by the immunoperoxidase method.13 The 2 actin specific, primary antibodies were diluted 1:200.
Cells were stimulated with AngII (10−7mol/L), AT1-RAb (dilution 1:40, titre detected by ELISA). Before the studies were performed, all cells had been maintained in Dulbecco's Modified Eagle's medium (DMEM) without new born serum for 24 hours. Cell density was not less than 105/cm2.
Cell proliferation assessment
VSMCs proliferation was determined by bromodeoxyuridine (BrdU) Cell Proliferation Assay Kit. BrdU was added to the cells in the six groups at different times (free serum group, Ang II group, AT1-RAb group, AT1-RAb with PDTC group, AT1-RAb with AG490 group, AT1-RAb with losartan group). Cell samples were harvested after 24 hours of incubation, washed and fixed as described in the instruction. ELISA assays for the incorporated BrdU were performed as described in the kit protocol. Briefly, the DNA in fixed cells was first denatured in 200 μl denaturing solution at room temperature for 30 minutes. Then detector anti-BrdU monoclonal antibody was added and incubated at room temperature for 1 hour, followed by incubating with horseradish peroxidase conjugated goat antimouse IgG for another 30 minutes. The horseradish peroxidase catalyzed the conversion of the chromogenic substrate tetramethylbenzidine from a colourless solution to a blue solution, the intensity of which (450 nm) was proportional to the amount of incorporated BrdU in the cells. The coloured reaction product was quantified using a spectrophotometer.
After stimulation, cells were harvested and the total proteins were prepared. Western blotting was performed as described before.9 To reduce the experimental error; we detected the expression of JAK2 and phosphorylated-JAK2 (pJAK2) in the same nitrocellulose membrane. The membrane incubated with anti-pJAK2 antibodies, washed with stripping buffer (100 mmol/L 2-mercaptoethanol, 2% SDS, 62.5 mmol/L Tris-Cl pH 6.7), then blocked and incubated with anti-JAK2 antibodies and following steps were as for Western blotting. Anti-STAT1 and antiphosphorylation-STAT1 (pSTAT1), anti-STAT3 and antiphosphorylated-STAT3 (pSTAT3) were incubated in a membrane too. Each Western blot experiment was done 5 or more times. Representative examples are shown. Quantification of the protein bands was carried out by laser densitometry.
Electrophoretic mobility shift assay (EMSA)
After stimulation, cells were harvested and nuclear extract was obtained.14 Nuclear factors activities were measured by electrophoretic mobility shift assay. NF-κB consensus oligonucleotide (5-AGTTGAGGGGACTTTCCCAGGC-3). The double stranded, NF-κB oligonucleotide was labelled with biotin. Binding reactions were carried out for 20 minutes at room temperature in the presence of 50 ng/μl poly(dI-dC), 0.05% Nonidet P-40, 5 mmol/L MgCl2, 10 mmol/L EDTA and 2.5% glycerol in 1 ×binding buffer (LightShift™ chemiluminescent EMSA kit, Pierce) using 20 fmol of biotin, end labelled target DNA and 2 μg of nuclear extract. Unlabelled target DNA (4 pmol) was added to 20 μl of binding reaction where indicated. Assays were loaded onto native 12% polyacrylamide gels pre-electrophoresed for 60 minutes in 0.5×Tris borate/EDTA and electrophoresed at 100 V for 2.5 hours before being transferred onto a positively charged nylon membrane (Hybond™-N+) in 0.5 × Tris borate/EDTA at 380 mA for 30 minutes. Transferred DNAs were crosslinked to the membrane at 120 mJ/cm2 and detected using horseradish peroxidase conjugated streptavidin (LightShift™ chemiluminescent EMSA kit) according to the manufacturer's instructions.
The densities of protein bands were quantified and graphically represented as mean ± standard deviation. The data were analyzed with SPSS 10.0 and used analysis of variance (ANOVA) followed by SNK comparison. All experiments were replicated at least three times. Differences were considered significant at a value of P<0.05.
The expression of α2-actin in VSMCs
The positive cells, which expressed α2-actin proteins, were in brown in immunohistochemistry.15 Figure not shown.
The autoantibodies against AT1 receptor induced VSMC proliferation through JAK-STAT and NF-κB pathway
Figure 1 shows the effects of the autoantibodies against AT1-receptor (AT1-RAb) on VSMC proliferation assessed by BrdU incorporation during over 24 hours. Both AngII and AT1-RAb significantly stimulated proliferation within 12 hours compared to cells without being exposed to either AngII or AT1-RAb. Ang II induced proliferation exceeded AT1-RAb induced proliferation.
VSMCs were separately stimulated by AG490 (inhibitor of JAK2) and losartan prior to exposure to AT1-RAb. In response to AT1-RAb, VSMC proliferation was almost completely inhibited in the presence of losartan and partially inhibited in the presence of AG490 or PDTC. These results suggested that the VSMC proliferation stimulated by AT1-RAb was dependent on the activation of JAK-STAT signal transduction system.
Expression of JAK-STAT and NF-κB in VSMC via AT1 receptor induced by the autoantibodies against AT1 receptor
VSMCs were incubated with AT1-RAb for different times and lysates were subjected to Western blotting using polyclonal antibodies: anti-JAK2, anti-STAT1, anti-STAT3 and anti-pJAK2, anti-pSTAT1, anti-pSTAT3 and anti-NF-κB. The expression levels of nonphosphorylation of STAT1 and STAT3 had no obvious changes in 2 hours.
However, a pronounced increase in tyrosine phosphorylation of STAT3 protein was observed 5 minutes after the application of AT1-RAb. The phosphorylation of STAT3 rose sharply between 15 and 30 minutes, but fell after 60 minutes. The bands of pSTAT1 were weak and maximal at 30 to 60 minutes (Figure 2A). NF-κB was activated by AT1-RAb and there were two peaks at 15 and 120 minutes (Figure 2B). The activation of STAT3 response to AT1-RAb was more intense than STAT1 and the relative densities of the bands of pSTAT1/STAT1 and pSTAT3/STAT3 at peak time were 0.519±0.032 and 0.735±0.038 respectively. These data suggested that expression level of phosphorylation in STAT3 was significantly increased and higher than in STAT1 (SNK test, P<0.05). The densities of NF-κB/β-actin bands were 0.456±0.022 and 0.433±0.019 at 15 and 120 minutes (Figure 2C) and expression level was significantly higher than at other times (SNK-test, P<0.05).
The VSMCs were exposed to losartan, AG490 (a specific inhibitor of the JAK2 tyrosine kinase) and PDTC (a specific inhibitor of NF-κB) before AT1-RAb. Results showed that the expression of pJAK2, pSTAT1 and pSTAT3 were greater in Ang II and AT1-RAb groups than control, but were markedly weaker in losartan and AG490 groups (Figure 3A). Similarly, the expression of NF-κB was inhibited by losartan or PDTC (Figure 3B). The histogram showed the semiquantitative results of NF-κB expression and phosphorylation level about JAK-STAT. The AT1-RAb group had the greater phosphorylation compared with control and blocker groups, P<0.05 (Figure 3C). It seems that the phosphorylation of JAK2 induced by AT1-RAb was unlikely to be mediated by de novo synthesized proteins but directly by AT1 receptor, because it occurred quickly (within 5 minutes) after stimulation with the AT1-RAb. Besides, it indicated that the JAK2 played a specific role in the tyrosine phosphorylation of STAT1 and STAT3.
Nuclear factors activities in VSMC via AT1 receptor induced by the autoantibodies against AT1 receptor
In the electrophoretic mobility shift assay, AT1-RAb increased the mobility of the consensus oligonucleotides for NF-κB as did Ang II; an effect that was inhibited by losartan and by PDTC (Figure 4).
It was recognized that Ang II could act not only as a vasoactive peptide but also as a growth factor. In particular, Ang II stimulates proliferation and hypertrophy in VSMC via AT1 receptor binding.10 Our results demonstrated that AT1-RAb caused a significant increase in BrdU incorporation similar to Ang II during 0–24 hours. However, the effect on cell proliferation was weak in AT1- RAb group; and the reason might be that affinity of AT1 receptor to Ang II was stronger than that of AT1-RAb.
The activation of JAK induced by cytokines or growth factors led to the rapid phosphorylation of STAT proteins on tyrosine residues. The phosphorylated STAT proteins could translocate immediately to nucleus, bind to specific DNA sequences and hence activate specific gene expression as in downstream reaction via AngII.16–19 Western blotting suggests activation of JAK-STAT was mediated mainly via the rapid, JAK induced, tyrosine phosphorylation of STAT3 transcription factors. The effect could be inhibited by losartan and AG490. Therefore, we verified that the STAT3 via JAK2 dependent pathway was essential, which is different to Ang II mainly via JAK2-STAT1.11 NF-κB was activated with two peak times. The result of EMSA showed that the AT1-RAb promoted NF-κB translocation into the nucleus (low STAT1), which is in accord with the previous results. As Figure 1 shows, the proliferation was diminished by losartan and by AG490. It indicated that AT1-RAb stimulated proliferation in VSMC is via AT1 receptor and activation of JAK2 in its signal transduction like Ang II. These results indicated that JAK2-STAT3 and NF-κB were the main molecules stimulated by AT1-RAb.
Ang II plays an important role in cardiovascular diseases and mediating biological responses by JAK-STAT and NF-κB. Our studies showed the AT1-RAb had agonist activity similar to AT1 receptor such as Ang II; AT1-RAb may be an important molecule in cardiovascular disease. Liao et al20 found that the AT1-RAb was positive in 43% patients with refractory hypertension and 10.4% with nonrefractory hypertension. In addition, the AT1 receptor antibodies induced arterial structural changes9 and the AT1-RAb stimulated proliferation of VSMCs without desensitization of the AT1 receptor with time period.3 We deduced that AT1-RAb participated in the pathogenesis of hypertension with positive AT1-RAb and maintain blood pressure. It was likely to be a reason why blood pressure could not be manageable. However, it still needs further study. Many studies21,22 demonstrated that STAT and NF-κB were important proinflammatory molecules, so we suggested that vascular inflammation would happen in hypertension patients with positive AT1-RAb.
The antagonist of AT1 receptor blocks agonist activity of internal Ang II, downregulates the expression of AT1 receptor23 and obstructs the effect of the AT1-RAb. Clinically, we used the AT1 receptor antagonist in hypertensive patients with positive AT1-RAb. In addition, we found that blood pressures of these patients were easier to control than by ACEI.20 Whether immunoabsorption or other ways to remove autoantibodies were valid in hypertension should be further tested. Besides, AT1-RAb had been found in some healthy people,5 thus we should pay attention to these people if they are more susceptible to suffer from hypertension than the people without the autoantibodies are.
Our experiments demonstrated that AT1-RAb induced VSMC proliferation and activated a definite chain of signal transduction from the cell membrane to the nuclear transcriptional molecules: the JAK2-STAT (mostly STAT3) and NF-κB signalling molecules were activated and thus regulating the gene expression resulting in biological action. Figure 5 shows that the process is similar to the agonist like activity of Ang II: some of their effects are different. In addition, we are not sure whether STAT3 is necessary in other immunological mechanisms of hypertension via JAK2 dependent pathways.
The finding of a signal transduction mechanism involving AT1-RAb in VSMCs reveals the possibility of regulating the activity of JAK-STAT and NF-κB signal transduction molecules in the treatment of hypertension.
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Keywords:© 2008 Chinese Medical Association
autoantibody; angiotensin AT1 receptor; proliferation; Janus kinase-signal transduction; activation of transcription; nuclear factor-κB