The Prevention Effects Of Azithromycin Or/and Cilostazol On Intimal Hyperplasia And Their Mechanism After Angioplasty In Rabbits

Objective and backgroundInflammation and platelet may be very important in the progress of intimal hyperplasia, and the letter is the principle mechanism of restenosis after PTC A. But, little is known about the effect of antibiotic and it is used together with the antiplatelet medicine on this progress, as well as their mechanism. We investigated whether microlide antibiotic Azithromycin or/ and Cilostazol can inhibit intimal hyperplasia after angioplasty in rabbits, and we hypothesized that both of those two medicine play their role by inhibiting the inflammation and platelet, especially decrease the level of CPIgG antibody titer, serum CRP, as well as VCAM - 1mRNA express level. And this inhibitory role can influence the balance of MMPs and TIMPs through MAPK (p44 ERK1/p42ERK2) pathway.Materials and MethodsAnimal model:60 Japanese male white rabbits weighing 1. 8-2. 8kg(4-5 months old) were divided into five groups: normal control group: fed by nonnal diet; high cholesterol control group: fed by high cholesterol diet (1.5g/kg/day) ; Azithromycin treat group: high cholesterol diet + Azithromycin (30mg/kg/day) for three days before angioplasty, and then one time every 6 days for 4 weeks; Cilostazol treat group: high cholesterol diet + Cilostazol (50mg/kg/day) started at three days before angioplasty for 4 weeks; both medicine treat group: high cholesterol diet + Azithromycin + Cilostazol , the dose and the use as aboveAccording the dividing, rabbits were fed for 4 weeks, then the aortic endo-thelium was denuded by inflating and stretching balloon (4. 0mm) , then contin-uoued their original diet for another 4 weeks, and followed by abdominal aortic angioplasty. 3 days before angioplasty, rabbits were given Azithromycin, Cilostazol, or both of them individually. After these 3 days, angioplasty was carried out, if the lumen area decreased 50% assessing by angiography under X -ray. After finished the angioplasty, all rabbits were fed normal diet for 4 weeks before sacrifice.Sample collecting:Four weeks later, animals were anaesthetized by 846 mixture. The distal segments of abdominal arteries were harvested after perfusion fixed physiological pressure . Part of angioplasty site of arteries were immediately frozen in liquid nitrogen, and then stored at — 80℃ for reverse transcription polymerase chain reaction and western blot analysis. Parts of them were fixed for morphormetry and immunohistochemistry and then were dehydrated, embedded and serial cross section - cut for HE, VG and Verhoeff staining, and the vascular neointimal, medium, and cavity area were measured by computer image analysis system. Immunohistochemical analysis were performed by SABC method staining with MMP2, MMP9, TIMP1, TIMP2, ERK1/2 primary antibody. For negative control, primary antibodies were omitted.Arterial segment stored at - 80℃ were taken out, and extracted total RNA and protein. RT - PCR and Western Blot analysis were carried out as previous described procedure. RT - PCR results were adjusted for GAPDH mRNA levels. All results were analysis by computer image analysis system.Blood samples were collected from the ear artery at before be fed , the first time injury, given different medicine, and before death.Statistical analysis:Quantitative data are expressed as mean ± standard deviation, and analyzed by Student's t test . A value of P <0. 05 was considered significant.Results1. Serum lipid changes:The serum TC, HDL - C, LDL - C increases, while HDL - C/TC ratio decreased after high cholesterol diet, compared with basic levels and normal control group before the two times balloon injury. But 4 weeks after angioplasty, because normal diet was given, the TC, HDL - C, LDL - C, and HDL - C/TC were returned to the baseline level. There was no difference among three treatments groups and high cholesterol control group in the serum lipids levels . TG level remained unchangable in the process of experiment.2. The inhibitory effect of three treatments on intimal proliferation after angioplasty.Azithromycin or/and Cilostazol can reduced the neointimal area (NIA) and NIA/MA (media area) , compared with the high cholesterol control group. The MA of all groups was on the same level. The change of NIA by Azithromycin or/ and Cilostazol only account for 50 - 70% of the change of CA (cavity area). TEM showed that in high cholesterol control group, SMCs were synthesis pheno-type, while in three treat groups, SMCs were tended to transforming to the cont-rictive phenotype.3. The effects of Azithromycin or/and Cilostazol on the inflammation markers.First, the CPIgG antibody titer was increased in all groups after the first time injury, especially the high cholesterol control, but it was decreased by Azithromycin treatment at 4 weeks after angioplasty.Second, the serum CRP level was higher in all groups before angioplasty than that in normal control group. While using Azithromycin or/and Cilostazol for 4 weeks could reduce this level, the high cholesterol control group stayed high level.Third, the VCAM - 1 mRNA level were decreased by Azithromycin or/and Cilostazol treatment, compared with high cholesterol control group.4. The inhibitory effect of Azithromycin or/and Cilostazoi on the extracellular matrix turnover:MMP2, MMP9 mRNA levels in high cholesterol control group increased at 4 weeks after angioplasty than that in normal control. And Azithromycin or/and Cilostazoi can reduced these levels. Both medicine treat group decreases them more prominently.TIMP1, TIMP2 mRNA levels experienced the same change.The same results appeared in immunohistochemistry, all MMP2, MMP9, TIMP1, TIMP2 were expressed more extensively and intensely in high cholesterol control group than in normal control group. Azithromycin or/and Cilostazoi can decrease the positive area and intensity. Interestingly, the most intensely positive area was located near from the internal elastic lamina.5. The effect of Azithromycin or/and Cilostazoi on ERK1/2 signal tranduct pathway:The expressing level of ERK1/2 protein was analyzed by Western Blot and Immunohistochemistry, the results showed that, in high cholesterol group, ERK1/2 protein level was the highest, and Azithromycin or/and Cilostazoi can reduce those two protein levels. The most intense positive area was near from internal elastic lamina, either.DiscussionIn the present study, we found that Azithromycin or/and Cilostazoi can inhibit the intimal hyperplasia. Especially, Azithromycin and Cilostazoi used together can reduce neointimal formation more obviously. And the neointimal area they minimized only account for 50 - 70% the cavity area they increased, it shows that Azithromycin or/and Cilostazoi can inhibit the arterial remodeling after angioplasty. Among three treatments and high cholesterol control group, there is no difference in serum lipids level, so we concluded that, the role of Azithromycin or/and Cilostazoi has no relation with lipid metabolism.Azithromycin can reduce plasma CPIgG antibody titer, compared with high cholesterol control group and Cilostazoi treat group . And Azithromycin or/andCilostazol can decrease the serum level of CRP, as well as VCAM - 1 mRNA expression at the angioplasty site. The results demonstrated that Azithromycin or/ and Cilostazol can inhibit the inflammation response after angioplasty, Azithromycin may be play its role by its antichlamydia character,it can inhibit organism LPS and the following inflammation, and Cilostazol, a new antiplatelet medicine and a specific in hibitor of cyclic AMP phosphodi esterase Ⅲ, can improve the cAMP level and prevent the platelet from activating and releasing many growth factors such as PDGF, prevent those factors from leading to inflammation. Many data showed that Restenosis is a chronic inflammation progress after angioplasty, while Azithromycin or/and Cilostazol can relieve this response, and prevent the development of RS.But how the inflammation play its role, and what mechanism Azithromycin or/and Cilostazol inhibits intimal hyperplasia were unclear. In our study, the results showed that the matrix metabolism played very important role. Many scholars believed that there would be a plenty of extracellular matrix to be produced and deposit at late phase after PTCA, which may be the main reason of neointi-mal formation at that period. And the balance between MMPs and their inhibitor TIMPs control the matrix synthesis and degradation. MMP2, MMP9 are necessary for SMC immigration and secretion. Our results demonstrated that, at 4 weeks after angioplasty, MMP2, MMP9, TIMP1, TIMP2 mRNA and protein level at the injury site were higher in high cholesterol control group than in normal control group. Azithromycin or/and Cilostazol treatment can inhibit the expression of these enzymes involving matrix turnover. That indicated that matrix turnover play important role in neointimal formation after angioplasty, and Azithromycin or/and Cilostazol inhibited the intimal hyperplasia by regulating MMPs, TIMPs expression, they inhibited extracellular matrix synthesis and prompted their degradation, inhibited SMC immigration.In Addition, what pathway do those inflammation factor and growth factor initiate those proliferative and secretive events? Now, it is well accepted that the signal transduct pathway is the last common way, especially MAPKs pathway. Two of them, ERK1/ERK2, are necessary for SMC proliferation and transfor-...