The Effects Of ACE And PAs And TGF-β1 On Restenosis After Angioplasty: An Experimental Study

Objective (1) To evaluate the effect of captopril on restenosis in the rat common carotid artery injury model. (2) To investigate the dynamic changes of PAs in each layer of the artery wall during each phase. Then to conclude the expression and significance of PAs during restenosis. (3) To investigate the dynamic changes and distribution of TGF- β 1 in each layer of the artery wall. Then to deduce their expression and significance during restenosis.Materials and Methods1 .Animal models and groupingCommon carotid artery injury model were created in 60 Male Sprague-Dawley rats on one side by improved guidwire method. Captopril (10m g/k g/d p.o.) was administered through a gastric sonde for 6 days prior to injury until sacrifice. The contralateral side acts as control. Specimens on both sides were harvested at five time points that was 1st, 4rd, 7th, 14th and 28th day. There are six rats at each time point. According to the HE staining slices, 50 specimens whose structure was complete, neointima was obvious were selected, at least 5 specimens were involved at each time point in each group. At control group, 5 specimens were selected randomly. The 50 specimens were stained by following methods. 2.Pathological examinationAll specimens were stained with HE. TPA and PAI-1 were stained with immunohistochemistry. UPA and TGF- β 1 was stained with both immunohistochemistry (IHC) and in situ hybridization (ISH). 3.Pathological imaging analysisMorphological metry analysis were performed on HE slices. Positive cells number were counted in the neointima, media, and adventitia respectively for the uPA tPA PAI-1 TGF-β 1 IHC and uPA TGF- β 1 ISH slices.Results1.The effects of captopril on neointima hyperplasia and remodeling during restenosis afterangiography.1.1 Pathological changesAt lday following arterial injury, endothelial cells were denudated.except for the injury indication in the media, the thickness and area were decreased. 4 days after injury in the adventitia, the thickness and area were increased. At 7 days following injury, neointima can be seen;the thickness and area reached the peak in the media and in the adventitia.ECM was increased.The size of lumen was declinded. At 14 days following injury, the increased thickenness of neointima with stenosis of lumen can be seen. The amount of extracellular matrix increased.The adventitia was intact and the increased adventitial thickness was declined.The size of lumen decreased. 28 days following injury, neointima was stable. The lumen was stenosis obviously. The thickness and area back to normal in adventitia. The lumen surface was covered by endothelial.There were no significantly differences in the treatment group compared to the single injury group ,except for the neointimal area decreased obviously in the treatment group 14 and 28 days after injury.1.2 Changes of the intimal, medial and adventitial thickennessThe neointimal thickness increased gradually from the 7th day after injury and reached to the peak to 28 days after injury.There were no significantly change for both medial and adventitial thickness. There were also no significantly differences among the intimal,medial and adventitial thickness between the treatment group and the single injury group .1.3 Changes of the intimal, medial and adventitial areaThe neointimal area was increased gradually from the 7th day after injury and reached to the peak and was stable to the 28 days following injury. The neointimal area was significantly decreased in the treatment group compared to the single injury group at 14 days and 28 days following injury. There were no significantly differences for the medial and adventitial area between two groups.1.4 Change of stenosis ratio,EELA,IELA and vascular remodeling index(VRI)Stenosis ratio(SR) increased gradually during the development of restenosis and reached to the peak 28 days after injury.Compared with the single injury group, SR decreased significantly from the 14th to 28th day in the treatment group.There were no significantly differences for EELA,IELA and VRI in two groups at each time points. 2.The expression and significance of uPA^ tPA^ PAI—1 during restenosis 2.1 The expression and significance of uPA antigen and uPA mRNA during restenosisThe expression of uPA antigen can be seen in the vessel wall in control group. 1 day after injury,its expression present in the media and adventitia. At 4 days following injury,there were a small quantity of potitive cells in the neointima. At 7 days following injury ,lots of potitive cell can be seen in the neointima.The number of potitive cells further increased in the media and adventitia At 14 and 28 days following injury,the number of potitive cells decreased in the neotima, media and adventitia.The expression of uPA mRNA was similar to the expression of uPA antigen in the vascular wall. Except for the expression of uPA antigen significantlydecreased in the neointima at 28 days and in the media 7 days and the expression of uPAmRNA significantly decreased in the neointima 14 days. There were no obviously differences for the expression of uPA antigen and uPAmRNA in other time points in the vessel wall between two groups .There was no correlation among the expression of uPA antigen and the area or the thickness of the neotima, media and adventitia respectively.2.2 The expression and significance of tPA during restenosisA small quantity of potitive cells with tPA antigen can be observed in the control group. At 1 day after injury,we can see a small quantity of potitive cells in the media and adventitia. At 4 days after injury.there were a few of potitive cells with the tPA antigen in the neointima while the number of potitive cells increased in the media and adventitia. The number of potitive cells increased and reached to the peak at 7 days following injury in the neointima,medial and adventitia. The number of potitive cells decreased at 14 and 28 days following injury in the neotima, media and adventitia. A lot of potitive cells still can be seen in the neotima. There were no obviously differences for the expression of tPA antigen in each time points in the vessel wall between two groups.There was positive correlation among the expression of tPA antigen and the area of the neotima. There was no correlation among the expression of tPA antigen and the area of the media and adventitia respectively. There was positive correlation among the expression of tPA antigen and the thickness of the neotima and the media respectively.There was no correlation among the expression of tPA antigen and the thickness of the adventitia.2.3 The expression and significance of PAI— 1 during restenosisA small quantity of potitive cells with PAI— 1 antigen can be seen in the intima and media in the control group.At 1 day after injury, a small quantity of potitive cells can be seen in the media and can not be seen in the adventitia. At 4 days after injury.there were a few of potitive cells in the neotima and adventima while the number of potitive cells increased obviously and reached to the peak in the media. The number of potitive cells increased and reached to the peak at 7 days in the neotima. The number of potitive cells decreased distinctly in the media and adventitia.The number of potitive cells decreased at 14 and 28 days following injury in the neotima, media and adventitia. A lot of potitive cells still can be seen in the neotima.There were no obviously differences for the expression of PAI-1 antigen in other time points in the vessel wall between two groups ,except for the number of potitive cells decreasing distinctly in the neotima at 14 and 28 days following injury and in the media at 4 days in the treatment group.There was no correlation among the expression of PAI-1 antigen in the vessel wall and the area or the thickness of the neotima,media and adventitia respectively. There was no correlation among the expression of uPA antigen and which of PAI-1 antigen in the neotima and adventitia respectively.There was positive correlation among the expression of uPA antigen and the expression of PAI-1 antigen in the media, the expression of tPA antigen and the expression of PAI-1 antigen in the neotima,media and adventitia respectively. 3.The expression and significance of TGF- P 1 during restenosisA small quantity of potitive cells with TGF- P 1 mRNA can be seen in the adventitia and media while there was no positive cell in the intima in the control group. At 4 days after injury,the number of potitive cells increased in the adventitia and media while there was still no positive cell in the intima. At 7 days following injury ,a small quantity of potitive cells can be seen in the neointima, Peak occurred in the media. At 14th and 28th days following injury, the number of positive cells dropped gradually to baseline in the adventitia and media, the number of positve stained cells reached to the peak in the neointima at 28 days following injury. There were no obviously differences for the expression of TGF- ft 1 mRNA in other time points in the vessel wall between two groups except for the number of positive cells decreased obviously in the neointima at 28 days . There was no correlation among the expression of TGF- ft 1 mRNA and the area or the thickness of the neotima,media and adventitia respectively.The expression of TGF- ft 1 antigen present was very low in each time points in the vessel wall. May be TGF- ft 1 antigen was destroyed and losed during specimen operation.Conclusionl.Captopril decreased the neointimal area and the stenosis ratio after injury and inhibited the development of the neointima,but did not change the EELA and IELA and VRI and vascular remodeling.2.The expression of tPA^ uPA and PAI~ 1 antigen and uPAmRNA increased with the increase of the neointimal area. So they are involved in the development of restenosis. UPA antigen and uPA mRNA expression was in the media before neointimal development and was in the neointima after neointimal development. UPA probably involved in the migration of proliferated cells toward intima during the early phase. The peak of tPA and PAI-1 antigen expression was in the late phase after neointima development. TPA and PAI-1 probably involved in the formation of neointima and vascular remodeling during late phase. There was positive correlation among the expression of tPA and PAI-1 antigen.maybe it is the compensed response of body to maintain the balance of PAs.3.At early phase following injury,TGF- ft 1 mRNA expression was primarily in the media. TGF-ft 1 mRNA expression was in the neointima after neointima development and the phase of TGF- ft 1 mRNA expression was very long. It showed that TGF- ft 1 mRNA involved in the formation of neointima during the late phase and vascular remodeling during restenosis. TGF- ft 1 antigen expression in vessel wall was very low at each time points . May be TGF- ft 1 antigen was destroyed and losed during specimen operation.