雌二醇抑制动脉球囊损伤后平滑肌细胞增殖
作者：赵智深 黄从新 江洪 王滕 王晶 李庚山
单位：湖北医科大学第一附属医院心内科，武汉 430060
关键词：雌二醇内膜增殖平滑肌内皮损伤颈动脉
中国临床药理学与治疗学000303
目的 探讨雌二醇对去势大鼠颈总动脉球囊损伤后内膜增殖的影响。方法 8～10wkSD大鼠雌性(n=21)、雄性(n=21)，各分3个组：非去势对照组(n=7)，去势对照组(n=7)，实验组(n=7，去势+雌二醇)。腹腔注射雌二醇3d后，2.0FPTCA球囊损伤左颈总动脉。损伤2wk后处死大鼠，测量内膜面积和中膜面积、内膜面积与中膜面积的比值。结果 雄性实验组内膜面积(0.072±0.020)mm2、内膜面积与中膜面积的比值0.533±0.037均显著小于非去势对照组(0.110±0.018)mm2，0.740±0.051,P均<0.01，也均小于去势对照组(0.098±0.014)mm2，0.701±0.040,P均<0.05。雌性实验组内膜面积(0.061±0.015)mm2、内膜面积与中膜面积比值0.525±0.030均显著小于去势对照组(0.101±0.018)mm2,0.710±0.031,P均<0.01，但与非去势对照组(0.078±0.012)mm2、0.619±0.041差异不显著，P均>0.05。结论 雌二醇能够抑制去势大鼠动脉损伤后内膜增殖。
, http://www.100md.com
中图分类号 R364.3
Estradiol in inhibition of proliferation of vascular smooth muscle cells after balloon injury
ZHAO Zhi-Sheng，HUANG Cong-Xin，JIANG Hong，WANG Teng，WANG Jing，LI Geng-Shan
(Department of Cardiology, First Affiliated Hospital of Hubei Medical University, Wuhan 430060)
Aim To investigate estradiol inhibition of neointimal proliferation after rat carotid artery balloon injury. Methods Eight to ten-week-old SD rats (male,n=21,female,n=21) were divided into intact control(n=7),gonadectomy control(n=7) and estradiol (n=7, gonadectomy)groups in each sex. Left carotid artery was not injured with 2.0 F PTCA balloon until estradiol was injected for three days. Rats were killed 2 wk after injury. Neointimal areas and media area, ratios of intimal areas/media areas were measured with computer. Results Male neointimal areas and ratios of intimal areas /media areas in estradiol group were less than those in intact control group significantly(all P<0.01) and than those in gonadectomy control group (all P<0.05). Those in female,in estrdiol group were less than those in gonadectomy control group evidently (all P<0.01),and similar to those in intact control group (all P>0.05). Conclusions Estrdiol inhibits neointimal proliferation after the gonadectomy in rats undergoing carotid artery balloon injury.
, http://www.100md.com
Key words estradiol； neointimal proliferation； smooth muscle cells； vascular endothelial injury; cartoid
In the thirties of the twentieth century , among 40～ 60 year-old human beings the morbidities of atherosclerosis and coronary heart disease(CHD) were higher in men than in women,or there was a sexual difference in cardiovascular diseases significantly.Then the interesting phenomenon was not considered to be related with estrogen.Godsland[1] and Knopp[2] in 1980's noted that the morbidity of CHD in women who subjected to ovariectomy with estrogen replacement treatment was much lower than that of whom without estrogen. Stamler J et al[3] first confirmed the fact that estrogen could hinder the development of atherosclerosis in chick fed with high cholesterol food. Afterwards, much more e- strogen tests were carried out and eventually estrogen protective effect on woman cardiovascular system was recognized. It may decrease plasma lipids and lipoprotein, increase high density lipoprotein cholesterol, and improve vascular endothelium. But its explicit mechanism was incompletely understood. Although 50% ～ 80% of patients with CHD can be cured with percutaneous transluminal coronary angioplasty, the procedure has been perplexed with following restenosis. Aim of this study was to observe whether estradiol inhibited vascular smooth muscle cells(VSMCs) proliferation after artery balloon injury.
, http://www.100md.com
1 Material and methods
1.1 Animals 8～ 10 week-old SD rats [male, n=21, weighing(341± 20)g; female, n=21, weighing (240± 17)g]were obtained from Exper imental Animal Center of Hubei Medical University. Rats with both sex were divided into three groups: intact control (IC, n=7), gonadectomy control(GC,n=7) and test groups (n=7,gonadectomy plus estradiol). Estradiol was purchased from Shanghai Ninth Pharmaceutical Factory (Lot № 890902111). Artery cross-sections were measured with computer.
, http://www.100md.com
1.2 Gonadectomy Rats in IC groups and test groups were gonadectomized after anesthetization with 2% pentobarbital sodium 40 μ g· g- 1. Antibiotics was used for two (male) or three days (female).
1.3 Administration Three (male) or four days (female) after gonadectomy, the rats in test groups were injected with estradiol 20 μ g· g- 1· d- 1 ip for two weeks. Then the rats were killed. The rats in female test group were given estradiol one day later than the male as to clear up the effect of intrinical estrogen on the study.
, 百拇医药
1.4 Arterial intima injury The method described by Chen et al[4] was consulted with some revision. A 2.0 F PTCA balloon catheter was introduced through the left external carotid artery and advanced into the thoracic aorta in aseptic manipulation.The balloon was inflated with 0.9% saline 0.2 ml and was pulled back to the bifurcation of common carotid artery. After the three times repeat of this procedure, the endothelium was almost removed. After the removal of the balloon, the external carotid artery was ligated and the wound closed. The right carotid artery was not damaged and served as a control. Rats in IC groups were injured 1 day after group division, in male GC group and male test group 3 days after gonadectomy and in female GC group and female test group 4 days after gonadectomy,were injured respectively.
, http://www.100md.com
1.5 Morphometric analysis Left and right common carotid arteries were isolated and rinsed with 0.9% saline ,then fixed in 10% formalin for 24 hours .The arteries were embeded in paraffine, sectioned and stained with HE. Cross- section neointima areas(=internal elastic lamina area-lumen area) and media area (=external elastic lamina area-internal elastic lamina area),ratios of neointimal areas/media areas were measured with computer.
1.6 Statistical analysis Data were expressed as
± s.Values of test groups were compared with those of IC groups and GC groups respectively by t test.
, http://www.100md.com
2 Result
Three rats died before they were killed: one in female GC group, two in female test group. Balloon injury on the endothelium was not equal so that the thickness of the neointimal proliferation was less consistent.Therefore the neointimal areas were taken as the extent of the intima proliferation. Ratios of intima area /media area were related neointimal formation with VSMCs hyperplasia. Intimal proliferation in male rats was seen in the Tab 1. Neointimal areas in test group were reduced by 30% compared with that in IC group [0.072± 0.02)mm2 vs (0.110± 0.018)mm2, P<0.01], they were also less than those in GC group [(0.098± 0.014)mm2, P<0.05]. Ratios of intima/media were also seen in Tab 1.
, 百拇医药
Tab 1 Left carotid artery intimal areas,ratios of intima areas/media areas in male rats(± s,n=7) Groups
Intima areas /mm2
Intima/media
IC
0.110±0.018
0.740±0.051
GC
0.098±0.014*
0.701±0.040
, 百拇医药
Test
0.072±0.020**＃
0.533±0.037**
compared with IC group* P<0.05,* * P<0.01;compared with GC group ＃ P<0.05
Intimal proliferation in female rats was seen in the Tab 2. Neointimal areas in test group were reduced by 40% compared with GC group [(0.061± 0.015)mm2 vs (0.101± 0.018)mm2,P<0.01], but it was less than that in IC group. Ratio of intima/media in test group 0.525± 0.030 was less significant compared with that in IC group (0.619± 0.041, P<0.05) and GC group (0.710± 0.031, P<0.01).
, 百拇医药
Tab 2 Left carotid artery intimal areas,ratios of intima areas/media areas in female rats(± s) Groups
n
Intima areas /mm2
Intima/media
IC
7
0.078±0.012
0.619±0.041
, http://www.100md.com GC
6
0.101±0.018*
0.619±0.041*
Test
5
0.061±0.015#
0.525±0.030#
compared with IC group* P<0.05;compared with GC group ＃ P<0.05
3 Discussion
, http://www.100md.com
The present experiment demonstrated that, (1) intimal areas in two test groups were reduced by 30% ～ 40% compared with those in their corresponding GC groups, suggesting that either extraneous or intrinsic estrogen inhibited neointimal hyperplasia;(2) androgen promoted neointimal hyperplasia in some way.
In the present study estradiol reduction of 30% ～ 40% neointimal hyperplasia after artery injury was similar to that reported by Chen et al[4]. Akishita et al[5] observed that administration of estradiol for two weeks did not alter the rats blood pressure and plasma lipids. So we speculated that estrogen inhibition of neointimal formation after balloon injury was not the change of lipid. It was also demonstrated that endothelin-1 increment induced expression of c-fos oncogene and increased VSMCs DNA synthesis. Estradiol inhibited VSMCs hyperplasia after balloon injury by the decrease of endothelin-1 level. Song et al[7]found that estradiol, on cultured VSMCs, prolonged to the synthetic type from the contractile after SMCs stimulation. Tourneau et al[8] observed that most neointimal volume was composed of extracellular matrix produced by synthetic SMCs, and at restenosis-region of intimal hyperplasia there were SMCs with aboundant extracellular matrix. Taken together, the mechanism of estradiol inhibition of neointimal formation after injury was related with low of endothelin-1, reduction of c-fos oncogene, and prolongation of SMCs transformation by estrogen receptor. Finally reduction of neointimal formation and media thickness after artery injury enlarged arterial lumen area.
, 百拇医药
Estradiol incompletely inhibited neointimal formation and thickness of media in the study. That meant other factors on which estrogen had no effect affecting neointimal formation. Jenkins et al[8]and his coworkers noted that membrane metalloproteinase-2 raised early after intima injury, and later it was located at the area of neointimal hyperplasia. A rise in membrane metalloproteinase-2,type 4 collagenase, activity made VSMCs easily pass through basal membrane to intimal region and multiply.That partly explained that estradiol inhibited neointimal formation incompletely.
, 百拇医药
Estradiol might have some effect on reduction the menopausal women with restenosis after angioplasty. Mechanism of restenosis is quite complex, so that its treatment will be difficulty.
ZHAO Zhi-Shen,male,doctoral student,engaged in research of interventional cardiology.
Reference
1，Godsland IF, Wynn V, Crook D,et al. Sex, plasma lipoproteins, and atherosclerosis: prevailing assump-tions and outstanding questions. Am Heart J, 1987;114:1467
, http://www.100md.com
2，Knopp RH. The effects of postmenopausal estrogen on the incidence of arteriosclerotic vascular disease.Obstet Gynecol, 1988;72 (Supp25):23s 3 Stamler J,Pick R,Ratz LN. Prevention of coronary atherosclerosis by estrogen-androgen administration in cholesterol-fed chick. Circ Res,1953;1:94
4， Chen SJ, Li H, Durand J,et al. Estrogen reduce neointimal proliferation after balloon injury of rat carotid artery. Circulation, 1996;93:577
, 百拇医药 5，Akishita M, Ouchi Y, Miyoshi H,et al. Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vas-cular smooth muscle cells.Atherosclerosis, 1997;130:1
6，Akishita M, Ouchi Y, Mioshi H,et al. Estrogen inhibits endothelin-1 production and c-fos gene ex-pression in rat aorta. Atherosclerosis, 1996;125:27
7，Song J, Wan Y, Rolfe BE,et al. Effect of estrogen on vascular smooth muscle cells is dependent upon cellular phenotype. Atherosclerosis, 1998;140:97
, 百拇医药
8，Tourneau TL, Van Belle E, Corseaux D,et al. Role of nitric oxide in restenosis after experimental balloon angioplasty in the hypercholesterolemic rabbit: effects on neointimal hyperplasia and vascular remodeling. J Am Coll Cardiol, 1999;33:876
9，Indolfi C, Esposito G, Dilorenzo E. Smooth muscle proliferation is proportional to the degree of balloon injury in rat model of angioplasty. Circulation, 1995; 92:1230
2000- 05- 22 received， 2000- 07- 20 accepted, 百拇医药
单位：湖北医科大学第一附属医院心内科，武汉 430060
关键词：雌二醇内膜增殖平滑肌内皮损伤颈动脉
中国临床药理学与治疗学000303
目的 探讨雌二醇对去势大鼠颈总动脉球囊损伤后内膜增殖的影响。方法 8～10wkSD大鼠雌性(n=21)、雄性(n=21)，各分3个组：非去势对照组(n=7)，去势对照组(n=7)，实验组(n=7，去势+雌二醇)。腹腔注射雌二醇3d后，2.0FPTCA球囊损伤左颈总动脉。损伤2wk后处死大鼠，测量内膜面积和中膜面积、内膜面积与中膜面积的比值。结果 雄性实验组内膜面积(0.072±0.020)mm2、内膜面积与中膜面积的比值0.533±0.037均显著小于非去势对照组(0.110±0.018)mm2，0.740±0.051,P均<0.01，也均小于去势对照组(0.098±0.014)mm2，0.701±0.040,P均<0.05。雌性实验组内膜面积(0.061±0.015)mm2、内膜面积与中膜面积比值0.525±0.030均显著小于去势对照组(0.101±0.018)mm2,0.710±0.031,P均<0.01，但与非去势对照组(0.078±0.012)mm2、0.619±0.041差异不显著，P均>0.05。结论 雌二醇能够抑制去势大鼠动脉损伤后内膜增殖。
, http://www.100md.com
中图分类号 R364.3
Estradiol in inhibition of proliferation of vascular smooth muscle cells after balloon injury
ZHAO Zhi-Sheng，HUANG Cong-Xin，JIANG Hong，WANG Teng，WANG Jing，LI Geng-Shan
(Department of Cardiology, First Affiliated Hospital of Hubei Medical University, Wuhan 430060)
Aim To investigate estradiol inhibition of neointimal proliferation after rat carotid artery balloon injury. Methods Eight to ten-week-old SD rats (male,n=21,female,n=21) were divided into intact control(n=7),gonadectomy control(n=7) and estradiol (n=7, gonadectomy)groups in each sex. Left carotid artery was not injured with 2.0 F PTCA balloon until estradiol was injected for three days. Rats were killed 2 wk after injury. Neointimal areas and media area, ratios of intimal areas/media areas were measured with computer. Results Male neointimal areas and ratios of intimal areas /media areas in estradiol group were less than those in intact control group significantly(all P<0.01) and than those in gonadectomy control group (all P<0.05). Those in female,in estrdiol group were less than those in gonadectomy control group evidently (all P<0.01),and similar to those in intact control group (all P>0.05). Conclusions Estrdiol inhibits neointimal proliferation after the gonadectomy in rats undergoing carotid artery balloon injury.
, http://www.100md.com
Key words estradiol； neointimal proliferation； smooth muscle cells； vascular endothelial injury; cartoid
In the thirties of the twentieth century , among 40～ 60 year-old human beings the morbidities of atherosclerosis and coronary heart disease(CHD) were higher in men than in women,or there was a sexual difference in cardiovascular diseases significantly.Then the interesting phenomenon was not considered to be related with estrogen.Godsland[1] and Knopp[2] in 1980's noted that the morbidity of CHD in women who subjected to ovariectomy with estrogen replacement treatment was much lower than that of whom without estrogen. Stamler J et al[3] first confirmed the fact that estrogen could hinder the development of atherosclerosis in chick fed with high cholesterol food. Afterwards, much more e- strogen tests were carried out and eventually estrogen protective effect on woman cardiovascular system was recognized. It may decrease plasma lipids and lipoprotein, increase high density lipoprotein cholesterol, and improve vascular endothelium. But its explicit mechanism was incompletely understood. Although 50% ～ 80% of patients with CHD can be cured with percutaneous transluminal coronary angioplasty, the procedure has been perplexed with following restenosis. Aim of this study was to observe whether estradiol inhibited vascular smooth muscle cells(VSMCs) proliferation after artery balloon injury.
, http://www.100md.com
1 Material and methods
1.1 Animals 8～ 10 week-old SD rats [male, n=21, weighing(341± 20)g; female, n=21, weighing (240± 17)g]were obtained from Exper imental Animal Center of Hubei Medical University. Rats with both sex were divided into three groups: intact control (IC, n=7), gonadectomy control(GC,n=7) and test groups (n=7,gonadectomy plus estradiol). Estradiol was purchased from Shanghai Ninth Pharmaceutical Factory (Lot № 890902111). Artery cross-sections were measured with computer.
, http://www.100md.com
1.2 Gonadectomy Rats in IC groups and test groups were gonadectomized after anesthetization with 2% pentobarbital sodium 40 μ g· g- 1. Antibiotics was used for two (male) or three days (female).
1.3 Administration Three (male) or four days (female) after gonadectomy, the rats in test groups were injected with estradiol 20 μ g· g- 1· d- 1 ip for two weeks. Then the rats were killed. The rats in female test group were given estradiol one day later than the male as to clear up the effect of intrinical estrogen on the study.
, 百拇医药
1.4 Arterial intima injury The method described by Chen et al[4] was consulted with some revision. A 2.0 F PTCA balloon catheter was introduced through the left external carotid artery and advanced into the thoracic aorta in aseptic manipulation.The balloon was inflated with 0.9% saline 0.2 ml and was pulled back to the bifurcation of common carotid artery. After the three times repeat of this procedure, the endothelium was almost removed. After the removal of the balloon, the external carotid artery was ligated and the wound closed. The right carotid artery was not damaged and served as a control. Rats in IC groups were injured 1 day after group division, in male GC group and male test group 3 days after gonadectomy and in female GC group and female test group 4 days after gonadectomy,were injured respectively.
, http://www.100md.com
1.5 Morphometric analysis Left and right common carotid arteries were isolated and rinsed with 0.9% saline ,then fixed in 10% formalin for 24 hours .The arteries were embeded in paraffine, sectioned and stained with HE. Cross- section neointima areas(=internal elastic lamina area-lumen area) and media area (=external elastic lamina area-internal elastic lamina area),ratios of neointimal areas/media areas were measured with computer.
1.6 Statistical analysis Data were expressed as
± s.Values of test groups were compared with those of IC groups and GC groups respectively by t test., http://www.100md.com
2 Result
Three rats died before they were killed: one in female GC group, two in female test group. Balloon injury on the endothelium was not equal so that the thickness of the neointimal proliferation was less consistent.Therefore the neointimal areas were taken as the extent of the intima proliferation. Ratios of intima area /media area were related neointimal formation with VSMCs hyperplasia. Intimal proliferation in male rats was seen in the Tab 1. Neointimal areas in test group were reduced by 30% compared with that in IC group [0.072± 0.02)mm2 vs (0.110± 0.018)mm2, P<0.01], they were also less than those in GC group [(0.098± 0.014)mm2, P<0.05]. Ratios of intima/media were also seen in Tab 1.
, 百拇医药
Tab 1 Left carotid artery intimal areas,ratios of intima areas/media areas in male rats(± s,n=7) Groups
Intima areas /mm2
Intima/media
IC
0.110±0.018
0.740±0.051
GC
0.098±0.014*
0.701±0.040
, 百拇医药
Test
0.072±0.020**＃
0.533±0.037**
compared with IC group* P<0.05,* * P<0.01;compared with GC group ＃ P<0.05
Intimal proliferation in female rats was seen in the Tab 2. Neointimal areas in test group were reduced by 40% compared with GC group [(0.061± 0.015)mm2 vs (0.101± 0.018)mm2,P<0.01], but it was less than that in IC group. Ratio of intima/media in test group 0.525± 0.030 was less significant compared with that in IC group (0.619± 0.041, P<0.05) and GC group (0.710± 0.031, P<0.01).
, 百拇医药
Tab 2 Left carotid artery intimal areas,ratios of intima areas/media areas in female rats(
± s) Groupsn
Intima areas /mm2
Intima/media
IC
7
0.078±0.012
0.619±0.041
, http://www.100md.com GC
6
0.101±0.018*
0.619±0.041*
Test
5
0.061±0.015#
0.525±0.030#
compared with IC group* P<0.05;compared with GC group ＃ P<0.05
3 Discussion
, http://www.100md.com
The present experiment demonstrated that, (1) intimal areas in two test groups were reduced by 30% ～ 40% compared with those in their corresponding GC groups, suggesting that either extraneous or intrinsic estrogen inhibited neointimal hyperplasia;(2) androgen promoted neointimal hyperplasia in some way.
In the present study estradiol reduction of 30% ～ 40% neointimal hyperplasia after artery injury was similar to that reported by Chen et al[4]. Akishita et al[5] observed that administration of estradiol for two weeks did not alter the rats blood pressure and plasma lipids. So we speculated that estrogen inhibition of neointimal formation after balloon injury was not the change of lipid. It was also demonstrated that endothelin-1 increment induced expression of c-fos oncogene and increased VSMCs DNA synthesis. Estradiol inhibited VSMCs hyperplasia after balloon injury by the decrease of endothelin-1 level. Song et al[7]found that estradiol, on cultured VSMCs, prolonged to the synthetic type from the contractile after SMCs stimulation. Tourneau et al[8] observed that most neointimal volume was composed of extracellular matrix produced by synthetic SMCs, and at restenosis-region of intimal hyperplasia there were SMCs with aboundant extracellular matrix. Taken together, the mechanism of estradiol inhibition of neointimal formation after injury was related with low of endothelin-1, reduction of c-fos oncogene, and prolongation of SMCs transformation by estrogen receptor. Finally reduction of neointimal formation and media thickness after artery injury enlarged arterial lumen area.
, 百拇医药
Estradiol incompletely inhibited neointimal formation and thickness of media in the study. That meant other factors on which estrogen had no effect affecting neointimal formation. Jenkins et al[8]and his coworkers noted that membrane metalloproteinase-2 raised early after intima injury, and later it was located at the area of neointimal hyperplasia. A rise in membrane metalloproteinase-2,type 4 collagenase, activity made VSMCs easily pass through basal membrane to intimal region and multiply.That partly explained that estradiol inhibited neointimal formation incompletely.
, 百拇医药
Estradiol might have some effect on reduction the menopausal women with restenosis after angioplasty. Mechanism of restenosis is quite complex, so that its treatment will be difficulty.
ZHAO Zhi-Shen,male,doctoral student,engaged in research of interventional cardiology.
Reference
1，Godsland IF, Wynn V, Crook D,et al. Sex, plasma lipoproteins, and atherosclerosis: prevailing assump-tions and outstanding questions. Am Heart J, 1987;114:1467
, http://www.100md.com
2，Knopp RH. The effects of postmenopausal estrogen on the incidence of arteriosclerotic vascular disease.Obstet Gynecol, 1988;72 (Supp25):23s 3 Stamler J,Pick R,Ratz LN. Prevention of coronary atherosclerosis by estrogen-androgen administration in cholesterol-fed chick. Circ Res,1953;1:94
4， Chen SJ, Li H, Durand J,et al. Estrogen reduce neointimal proliferation after balloon injury of rat carotid artery. Circulation, 1996;93:577
, 百拇医药 5，Akishita M, Ouchi Y, Miyoshi H,et al. Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vas-cular smooth muscle cells.Atherosclerosis, 1997;130:1
6，Akishita M, Ouchi Y, Mioshi H,et al. Estrogen inhibits endothelin-1 production and c-fos gene ex-pression in rat aorta. Atherosclerosis, 1996;125:27
7，Song J, Wan Y, Rolfe BE,et al. Effect of estrogen on vascular smooth muscle cells is dependent upon cellular phenotype. Atherosclerosis, 1998;140:97
, 百拇医药
8，Tourneau TL, Van Belle E, Corseaux D,et al. Role of nitric oxide in restenosis after experimental balloon angioplasty in the hypercholesterolemic rabbit: effects on neointimal hyperplasia and vascular remodeling. J Am Coll Cardiol, 1999;33:876
9，Indolfi C, Esposito G, Dilorenzo E. Smooth muscle proliferation is proportional to the degree of balloon injury in rat model of angioplasty. Circulation, 1995; 92:1230
2000- 05- 22 received， 2000- 07- 20 accepted, 百拇医药