Effect of valsartan-eluting stents on the expression of angiotensin II type 2 receptor

Percutaneous coronary intervention (PCI) with drug-eluting stent implantation has been proved efficient to reduce complications and restenosis in type B2/C lesions compared with bare stent, since the major drawback of the latter technique is intimal hyperplasia from the vessel media, which significantly cause in-stent restonosis. Despite the beneficial effects on the renin-angiotensinaldosterone (RAAS) system, the role of angiotensin-converting enzyme (ACE) inhibitors after PCI is still unclear. However, first results of angiotensin receptor antagonist (valsartan) after stent implantation in the Val-PREST1 and VALVACE2 trials have indicated the systemic pharmacological effect in the prevention of in-stent-restenosis. Up to our knowledge, whether local treatment of valsartan by implanting drug-eluting stent can generally prevent restenosis by inhibiting intimal hyperplasia has not been explored. The present study aimed to investigate the local efficacy of valsartan-eluting stent in inhibiting vessel intimal hyperplasia in comparison with the carrier-eluting as well as bare mental stent in rabbits' aortas in short term after implantation so as to explore meaning and possibility of the potential clinical application of valsartan-eluting stent in order to reduce the incidence of in-stent-restnosis in the future. METHODS Stents preparation Thirty stainless steel stents (316L Microport Co. China) with length 10 mm and diameter 3.0 mm were chosen, among which 20 stents were manufactured into eluted, i.e. 10 valsartan-eluting stents in which valsartan were embedded in base coating in a dosage of 300 μg/cm2; and another 10 carrier-eluting stents, by using the exactly same multi-layers coated technique, while 10 were left to be bare mental stents. According to the multi-layers coated technique (patent application No. CN 02136332.2), the eluted stents were manufactured with base coating, which was contented with organic-inorganic compound, and top coating, which was made with high compatible polyer full of phospholipid groups. The only difference of the two eluted stents was that valsartan was embedded in base coating during the manufacture in the valsartan-eluting stent, whereas the carrier-eluting stent was not. Animals and methods Fifteen adult New Zealand white rabbits (ignorance of gender, weighting 2.75 – 3.25 kg), were acclimatized to the animal quarters for at least one week. Thereafter they were randomized into three groups (5 per group): (1) bare stents group; (2) carrier-eluting stents group; (3) valsartan-eluting stents group, in which they were implanted two stents into celiac aorta of each rabbit with the three types stent as described following. Briefly, the animals were anesthetized celiacly with ketamine, the right common femoral artery was surgically exposed and the adventitia circumferentially cleared. A 4F arterial sheath was introduced through the incision. Meanwhile, heparin in a dosage of 100 IU/kg was injected through the sheath. An angioplasty balloon (3.0 mm×20 mm) with stent was introduced by a guide wire [0.0356 cm (0.014″)×190 cm] in the superficial femoral artery and advanced into the common femoral artery to a standardized location just distal to the bifurcation of the renal artery. The balloon was inflated to 1215.9-1418.6 kPa (12-14 atm) in 30-60 seconds before it was withdrawn and releasing the stent. Another stent was implanted with the same method and the distance between two stents was about 2 cm. The catheter was then removed, and the incision was closed after the implantation. Regular injection of 400 000 U benzylpenicillin sodium was administered for 3 days for prevention of infection. Quantitative angiography (QA) was performed before, immediately after, and 3 months after stents implantation respectively. Accordingly the vascular diameters of the aortas as well as the indices of the vascular neointimal formation, i.e. luminal area (LA), neointimal area (NIA), inner elastic membrane luminal area (IELA) and the maximal innermembrane thickness (MIT) were measured and compared thereafter in three groups. Three months later, the rabbits were executed. The vessel embedded stents were taken out and put into formalin (4%) for fixation for 24 hours, then was embedded into plastic. Slices were made by polycut slicemachine and HE stained. The HE stained slices images were analyzed by Leica Q550CW system. Immunohistochemical staning was performed on formalin-fixed, paraffin embedded sections by envision technique using the kit (Dako Corporation, USA) and anti-AT2 receptor antibody (Santa Cruz, USA). Reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used in the gene polymorphism analysis. Total RNA was extracted from arterial sample through the use of TRIzol reagent. Primer was designed with PRIMER5 software, AT2: 5′-AGGTTTCCAGCATTTACATC-3′; 3′-CTATCGCAACCGACCACTG-5′. β -actin: 5′-AG GAAGGAGGGCTGGAACA-3′; 5′-CCCATCTACGA GGGCTACGC-3′. Reaction condition was as follows: pre-denaturalization at 95°C for 5 minutes, denaturalization at 94°C for 40 seconds, annealing at 58°C for 40 seconds, extension at 72°C for 40 seconds, after 35 circles delay for 10 minutes at 72°C, agarose gel electrophoresis of PCR production was performed. Statistical analysis All data were analyzed by SPSS12.0 software. Descriptive data were expressed as mean ± standard deviation (SD). Analysis of variance (ANOVA) was used in comparison between the groups. Statistic significance was set at a level of P<0.05. RESULTS One rabbit died during the procedure of implantation, another one died of diarrhea during acclimatization. Therefore, there were 8 bare stents successfully implanted into 4 rabbits, and another 10 carrier-eluting stents and 10 valsartan-eluting stents into 5 rabbits respectively. Quantitative angiography There was no difference between any two of the three groups in terms of the mean diameters of the celiac aortic at before, immediately after or 3 months after implantation by QA comparison. No in-stent restenosis (diameter stenosis >50%) was found in any group according to the repeated QA at 3 months after implantation. Morphometric analysis It was indicated that a larger LA and less neointimal hyperplasia was significantly found in valsartan-eluting stent group compared to the other two groups. The average LA was (4 345 548±125 822) μm2, (4 302 061±167 952) μm2 and (5 016 269 ±207 934) μm2 at bare stent group, carrier-eluting stent group and valsartan-eluting stent group 3 months after implantation. It was also found that the MIT in all three group was significantly increased at 3 months after implantation. Furthermore, MIT and NIA in valsartan-eluting group were significantly smaller than those of the other two groups with a significantly larger LA as indicated in Table.Table: Results of HE staining slice in bare stent group, carrier-eluting stent group and valsartaing-eluting stent group (mean±SD)Results of immunohistochemistry The angiotensin II type 2 (AT2) receptor was found mainly in adventitia in bare stent group and carrier eluting stent group, while in adventitia and intima in valsartan-eluting stent group (Fig. 1).Fig. 1.: Expression of AT2 receptor (brown staining) in bare stent, carrier-eluting stent and valsartan-eluting stent groups (HE, original magnification × 200). A: bare stent; B: carrier-eluting stent; C: valsartan-eluting stent.Results of RT-PCR The mRNA of AT2 receptor is 404 bp RT-PCR product, and the mRNA of β-actin is 311 bp as shown in Fig. 2. The mRNA of AT2 receptor expressed significantly higher in valsartan-eluting group than in bare stent group and carrier-eluting stent group by RT-PCR.Fig. 2.: Expression of mRNA for AT2 receptor in bare stent group,carrier-eluting stent group and valsartan-eluting stent group.DISCUSSION It has been proved that stents could counteract aortic recoil and remodeling, so that in-stent restenosis almost is caused by intimal proliferation.3 To inhibit neointimal formation and reduce in-stent restenosis, there are two kinds of drug-eluting stents used clinically: rapamycin-eluting stent and paclitaxeleluting stent. Both rapamycin and paclitaxel are strongly anti-proliferative agents, which can inhibit smooth muscle cell (SMC) proliferation and migration so as to inhibit neointimal formation and reduce restenosis. However, late thrombosis, inflammation, hypersensitivity reactions and endothelium healing delay are of major concern. In light of the recent interest in more physiological endothelium and arterial recovering antirestenotic strategy, these findings to prevent in-stent restenosis; renin-angiotensin are worrisome system (RAS) intervention has endothelium- protective properties, even in the setting of vascular injury.4,5 VAL-PREST1 and VALVACE2 trials have proved that systemically administered AT1 receptor blockade could reduce in-stent restenosis. To study the effect of valsartan eluting-stents on vascular neointimal formation and AT2 receptor expression and access the feasibility to prevent restenosis, we coated stents with valsartan and carried out animals experiment. The results showed valsartan eluting- stents inhibited neointimal hyperplasia and might play an important role in preventing restenosis. The mechanism may be associated with the expression up-regulation of AT2 receptor mRNA in the wall of local artery. It is known that there are 4 subtype angiotensin II receptors: AT1, AT2, AT3 and AT4. RAS exerts its physiological effects through angiotensin II stimulating angiotensin II receptors, especially AT1 and AT2. Activation of the AT1 receptor has deleterious effects on the cardiovascular system, namely cell migration and proliferation, extracellular matrix deposition, inflammation, promotion of thrombosis, and production of reactive oxygen species. Activation of the AT2 receptor could counteract effects of the AT1 receptor activating. Activation of the AT2 receptor is associated with no production, inhibition of proliferation, and induction of apoptosis. Selective AT1 receptor blockers inhibit stimulation of AT1 receptor by angiotensin II, the AT2 receptor can still be activated. Adverse effects of angiotensin II, such as cell migration and proliferation, extracellular matrix deposition, inflammation, promotion of thrombosis, and production of reactive oxygen species, are blockaded, and the advantageous effects of AT2 activation still remain. AT1 receptor and AT2 receptor interact each other. Wu et al6 elucidated the function of the AT2 receptor during AT1 receptor antagonism after vascular injury. Neointimal formation was attenuated in wild mice by AT1 receptor blockade; this effect was less prominent in AT2 knockout mice. Moreover, anti-inflammatory effects of AT1 receptor blockade were decreased in AT2 knockout mice. Their conclusion was that stimulation of the AT2 receptor during AT1 receptor antagonism is important in the decrease of neointimal formation after vascular injury. Furthermore, Suzuki et al7 demonstrated neointimal formation as well as DNA synthesis in vascular smooth cells after vascular injury was exaggerated in AT2 receptor knockout mice, but they were both suppressed in AT1 receptor knockout mice compared with those in wild-type mice. In contrast, the number of apoptotic cells in the injured artery in vascular SMC was significantly increased in AT1 knockout mice but decrease in AT2 knockout mice. The results suggest AT2 exerts antiproliferative effects and propoptotic changes in vascular SMC by counteracting AT1 in the process of neointimal formation after vascular injury. In our study we found that the neointimal formation decreased and the expression of AT2 receptor mRNA was up-regulated in valsartan eluting-stent group, thus we believe that AT2 receptor played an important role in inhibiting neointimal formation. Our study showed that valsartan-eluting stents, local application of valsartan, could inhibit neointimal formation, and may be an attractive methods to prevent in-stent restenosis.