Preventive Effects And Its Mechanism Of Tetrandrine On Vascular Restenosis Induced By Intimal Denudation Of Carotid Arteries In Rabbit

Part IDynamic Model of Neointimal Proliferation and Remodeling of Vascular Wall after Balloon Intimal Injury of Rabbit Carotid Common ArteriesObjective: At present, many studies have reported that proliferation and migration of vascular smooth muscle cell ( VSMC ) in the media, and subsequent neointimal hyperplasia and remodeling of vascular wall is a major pathologic mechanism of vascular restenosis after injury of endothelial cells (intima ) resulted from percutaneous transluminal coronary angioplasty ( PTCA ). To study the mechanism of restenosis for the prevention and treatment following PTCA, we replicated the dynamic models of cellular proliferation and vascular remodeling after intimal denudation of rabbit carotid common arteries at different time points.Methods: The intima of left carotid common arteries in 42 rabbits was denudated by balloon injury. Rabbits were respectively killed after injury at different time points ( 1 d, 3d, 5 d, 7d, 14d, 28 d and 35 d ). The injured sections were taken out and made into specimens for HE staining. Morphological change of the arterial wall in injury sides and non-injury sides at different time points was observed by microscopy. In addition, Lumen area, thickness and area of the intima and the media, and cross-sectional area bounded by the external elastic lamina ( EELA) in injury sides and non-injury sides at different time points were measured by computer image analysis technology.Results: (1) Single layer endothelium was only showed in non-injury side arteries at different time points. Endothelial cells were denudated at day 1 after balloon injury. The proliferating of VSMC was spotted on the surface of lumen at 3 days after balloon injury. The neointima had been formed and continuously thicken at 5 ～7 days after injury. The neointimal thickness and area as well as extracellular matrix were gradually increased after 14 days, and they were maximal at 35 days. (2) The thickness and area of the media were gradually increased during 3-14 days, in which the medial area significantly increased at 14 days compared with non-injured vessel, and decreased after 28 days. Lumen area initially decreased at 5 ~ 7 days after injury. Lumen area after 14 days was significantly less than that of non-injured vessel. EELA gradually slightly increased at 1 ~ 7 days after injury, it was maximal at 14 days, but it declined gradually after 28 days.Conclusion: The models of vascular restenosis established by balloon intimal injury of rabbit carotid common arteries were successfully replicated, and proved to be rational and scientific. Intimal proliferation and vascular remodeling are important pathologic pathogenesis of vascular restenosis. The formation of vascular restenosis was determined by the unbalance of intimal proliferation and vascular remodeling. The both together resulted in lumen narrowing . Part II Experiment I Change of P38 MAKP and MKP-1 in Phenotypic Modulation of Vascular Smooth Muscle Cells after Intimal InjuryObjective: To explore relationship among phenotypic modulation of vascular smooth muscle cell ( VSMC ) and change of P38 mitogen-activated protein kinase ( P38 MAPK ) as well as mitogen-activated protein kinase phosphatase-1 ( MKP-1 ) after vascular intimal injury.Methods: The model of vascular restenosis established by balloon injury of rabbit carotid common arteries was used. Immunohistochemistry, western blot and reverse transcriptase-polymerase chain reaction (RT - PCR) were used to detect the change of proliferation cell nuclear antigen ( PCNA ), smooth muscle α - actin ( SMα - actin ), P38 MAPK protein, and MKP-1 protein as well as its mRNA in vascular wall of sham-injured group and injury group at different time points.Results: (1) Immunohistochemical staining of PCNA was negative in the medium and endothelium of sham-injured group. Positive cell percentage of PCNA was gradually increased at 1 ～14 days in the medium and at 5 ～14 days in the neointima after injury, and it reached peak value at 14 days. Positive cell percentage of PCNA was still higher though it was gradually declined after 28 days. Positive cell percentage of PCNA in the neointima was slightly more than that in the medium. (2) Immunohistochemical staining of SMα - actin was positive in the medium, negative in the endothelium of sham- injured artery. Positive expression area of SMα - actin in the medium initially decreased at 1 day and was minimal at 3 days after injury, but it increased gradually after 5 days, and was close to level of sham-injured artery at 28 days after injury. SMα -actin staining was always positive in the neointima at 5 ～35 days after injury, but its positive cells area was slightly less than that in the medium at different time points. (3) Immunohistochemical staining of P38 MAKP was negative or feeble positive in the medium of sham-injured artery. Positive expression area of P38 MAKP was significantly increased in the medium at 1 ～14 days and in the neointima at 5 ～14 days, and it was maximal at 14 days after injury. However, positive expression area of P38 MAKP initially decreased after 28 days, but its positive area was still significantly higher than that in sham-injured artery at 35 days after injury. P38 MAKP positive area in the neointima was higher than that in the medium. P38 MAKP examined by Western blot was sustained increased in vascular wall at 1～35 days after injury, in which it reached peak value at 14 days, initially decreased after 28 days. There were positive relationships between the change of P38 MAKP and PCNA in vascular wall at different time points after injury. (4) Immunohistochemical staining of MKP-1 was feeble positive or positive in the medium and endothelium of sham-injured artery. Its positive expression area in the medium progressing decreased at 1 ～3 days, and it reached lowest value at 5 ～ 7 days. At the same time, staining of MKP-1 was negative or feeble positive in the neointima. However, MKP-1 positive expression area was slightly increased from 14 to 28 days in the medium and the neointima. Its positive expression area was still lower than that sham-injured group at days 35 after injury. MKP-1 positive area in the neointima was slightly lower than that in the medium. The change of MKP-1 mRNA in vascular wall was consistent with immunohistochemical results. There were negative relationships between the change of MKP-1 and PCNA in the vascular wall at different time point after injury.Conclusion: There was close relationship between phenotypic modulation and proliferating ability of VSMC. P38 MAPK and MKP-1 could participate in phenotypic modulation of VSMC and its regulation after intimal injury.Part IIExperiment IIEffects of Tetrandrine on Phenotypic Modulation of Vascular Smooth Muscle Cells and Expression of P38 MAPK as well as MKP-1 after Intimal Injury of Rabbit Carotid ArteriesObjective: To study the effects of tetrandrine ( Tet ) on phenotypic modulation of vascular smooth muscle cells ( VSMC ) and P38 mitogen-activated protein kinase ( P38 MAPK) as well as mitogen - activated protein kinase phosphotase-1 ( MKP-1 ) after vascular intimal injury.Methods: The model of vascular restenosis established by balloon intimal injury of rabbit carotid common arteries was used. HE staining was used to analysis vascular morphology change of sham-injured, injured and Tet-treated group at 28 days. Immunohistochemistry, Western blot and RT - PCR were respectively used to detect the expression change of proliferation cell nuclear antigen ( PCNA ), smooth muscle α-actin ( SMα-actin), P38 MAPK and MKP-1 of vascular wall in injured group and Tet group at 7, 14 and 28 days after injury. Results: (1) The each layer structure in vascular wall was intact in sham-injured group arteries at day 28. The neointimal area was significantly increased and the lumen area notably decreased in injured group at day 28. Compared with injured-group, the neointima proliferation degree was evidently decreased, and lumen area was markedly increased in Tet treated-group at day 28. (2) Compared with injured-group, the expression of PCNA, SMα-actin, P38 MAPK and MKP-1 in vascular wall of Tet group had no difference and the neointimal proliferation degree was also basically same at day 7 after injury. The expression of PCNA and P38 MAKP in Tet group was all obviously lower than that in injured-group, whereas the expression of MKP-1 in Tet group was evidently higher than that in injured-group in vascular wall at days 14 and 28 after injury, and all had time dependent manner. The expression of SMα-actin in Tet group was slightly higher than that in injured-group at days 14 and 28 after injury. Conclusions: Tet could reduce neointimal proliferation by inhibiting phenotypic modulation and P38 MAPK signal transduction pathway as well as its negative regulation of VSMC after intimal injury.Part IIIExpressional Change of MMP9, TIMP2 and Effects of Tet on Their Expression in Vascular Wall after Intimal Injury of Rabbit Carotid Objective: To investigate (1) the effect of matrix metalloproteinases - 9 ( MMP9 ) and tissue inhibitors of metalloproteinases - 2 ( TIMP2 ) in extracellular matrix ( ECM ) formation of vascular restenosis; (2) the effects of tetrandrine (Tet) on MMP9, TIMP2 expression and vascular intimal proliferation. Methods: The model of restenosis established by balloon injury of rabbit carotid common arteries was performed. Immunohistochemistry, reverse transcriptase - polymerase chain reaction ( RT - PCR ) and Western blot were respectively used to detect the change of MMP9, TIMP2 mRNA and their protein expression in vascular wall as well as intimal proliferation in sham-injured group ( S group ) at 28 days, in injured group (I group ) and Tet-treated group ( Tet group ) at 7, 14 and 28 days after balloon injury. Results: (1) Positive expression area of MMP9 was higher in the medium at 7 days, and that it was gradually decreased at 14 and 28 days after injury; MMP9 positive expression area was lower relatively in the neointima at 7 days, and that it was significantly increased at 14 days, but it was obviously decreased at 28 days after injury. Positive expression area of TIMP2 in the medium and neointimal was lower at 7 days, and that it was significantly increased at 14 and 28 days after injury. (2) Compared with I group of same period, positive expression area of MMP9 and TIMP2 in the medium and neointimal of Tet group had no evident difference at 7 days, and that they were all respectively significantly decreased at 14 and 28 days after injury. At the same time, neointimal proliferation degree was also relieved and evidently relieved at 14 and 28 days after injury. (3) Expression of MMP9 mRNA and its protein was lower, and that the expression of TIMP2 mRNA and its protein was higher in vascular wall of S group. Compared with S group, expression of MMP9 mRNA and its protein in vascular wall of I group was stronger or higher at 7, 14, and 28 days. Expression of TIMP2 mRNA and its protein in vascular wall of I group was lower than that of S group at 7 days, and that it come back to or was higher than that of S group at 14 and 28 d. (4) Compared with I group of the same period, expression level of MMP9 mRNA and TIMP2 mRNA as well as their protein in vascular wall of Tet group had all no significant difference at 7 days, and that their expression of Tet group all had been significantly decreased at 14 days and 28 days after injury, in witch decrease of TIMP2 was more than that of MMP9. Conclusions: The lopsided increase and/or imbalance of MMP9 and TIMP2 expression played important role in ECM formation after vascular intimal injury. Tet could reduce abnormal higher expression of MMP9 and TIMP2 in vascular wall after intimal injury, ensure the equilibrium of MMP9 / TIMP2, and retard the accumulation and secretion of ECM, thereby inhibit neointimal hyperplasia and vascular remodeling.