Experimental study on induction of atherosclerotic plaque instability in rabbits after transfer of wild-type p53 gene

AIM: To study the apoptotic role of wild-type p53 in induction of plaque instability in atherosclerotic rabbits. METHODS: Fifty-four New Zealand White rabbits underwent balloon-induced abdominal aortic wall injury and then were fed on a diet of 1% cholesterol. At the end of the eighth week, the rabbits were randomly divided into two groups: group A and group B. Recombinant adenovirus carrying p53 and β-galactosidase (LacZ) genes were injected in group A and B, respectively. Two weeks later, 10 rabbits each in group A and B was killed and the remaining rabbits all underwent pharmacological triggering with injection of Chinese Russell's viper venom and histamine. RESULTS: Compared with group B, p53 gene over-expression in group A resulted in a marked increase in number of positive apoptotic cells (2.5%±0.8% vs 1.0%±0.3%, P<0.05) and a significant decrease in vascular smooth muscle cells (47.5%±6.8% vs 80.4%±10.6%, P<0.01), the thickness of the fibrous cap ［(132.9±56.7）μm vs （181.8±59.7） μm, P<0.05］ and the cap/intima-media ratio (0.20±0.18 vs 0.21±0.11, P<0.05). CONCLUSION: Transfer of human wild-type p53 genes effectively promotes apoptosis of VSMCs in atherosclerotic plaques, which makes the plaques vulnerable to rupture.     

Effect of human tissue factor pathway inhibitor gene transfection in vein grafts on thrombus formation and early patency rate

AIM: To reduce thrombus formation after coronary artery bypass graft,we investigated the antithrombotic effect of human tissue factor pathway inhibitor (TFPI) gene delivery on vein grafts. METHODS: The eukaryotic expressed plasmid vector pCMV-(Kozak)TFPI was constructed. Through pressurizing infusion,vein endotheliocytes were transfected with cationic liposome containing the plasmid vector pCMV-(Kozak)TFPI. After operation, vein grafts were harvested at third day for immunohistochemical, RT-PCR and Western blotting analysis of exogenous gene expression and for observation of thrombus formation by pathological method and scanning electron microscopy. At 30th days, the patency rate was recorded by vessel Doppler ultrasonography. RESULTS: Human TFPI mRNA and protein were detected in TFPI gene transferred vein grafts. Thrombosis was found in 8 animals of empty plasmid control group and in 7 animals of empty control group, but in only 1 of the TFPI group (P<0.05). Thirty days after operation, 5 vein grafts were occluded in both empty plasmid control group and empty control group, but none of vein grafts were occluded in TFPI group (P<0.05). The endothelial surfaces of the vein grafts in both control groups were covered with aggregated erythrocytes and platelets, but not in TFPI group. CONCLUSION: Human tissue factor pathway inhibitor gene transfection reduces thrombus formation and improves early patency rate in vein grafts.     

Inhibitory effect of recombinant human endostatin on angiogenesis in atherosclerotic plaque of rats by regulating Dll4/Notch pathway

AIM: To observe the inhibitory effect of recombinant human endostatin (rhES) on plaque angiogenesis, and to explore the regulatory mechanism of Dll4/Notch pathway in the anti-angiogenic effect of rhES. METHODS: Male Wistar rats were randomized into 3 groups:normal control group (N group), atherosclerotic model group (AS group), and rhES treated group (AS+rhES group). The rats in N group were fed a normal diet, while the remaining 2 groups were established to atherosclerotic rat model via high-cholesterol diet, intraperitoneal injection of vitamin D₃ and aortic balloon injury. The rats in AS+rhES group received intraperitoneal injection of rhES. The blood total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), C-reactive protein (CRP), interleukin-1 (IL-1) and troponin I (TnI) were measured. The atherosclerotic abdominal aortas were taken for pathological observation. Immunohistochemical staining was used to measure the density of neovessels in the plaques, which were marked by CD31. The protein levels of Dll4 and Notch1 in the aortas were analyzed by Western blot. RESULTS: The levels of blood TC, TG, LDL-C, CRP and IL-1 in AS group and AS+rhES group were much higher than those in N group (P<0.05), and no statistical difference between AS group and AS+rhES group was observed. The expression of CD31 in AS group was the highest among all groups. Compared with AS group, the density of neovessels in the plaques of AS+rhES group decreased significantly (P<0.05). The protein expression of Dll4 and Notch1 in AS group was lower than that in N group (P<0.05). Compared with AS group, the protein expression of Dll4 and Notch1 increased significantly (P<0.05). CONCLUSION: rhES has the ability to inhibit plaque angiogenesis in rats. The activation of Dll4/Notch pathway may be the mechanism of rhES in inhibiting plaque angiogenesis.     

Roles of PI3K/Akt/mTOR signaling pathway in macrophage autophagy and atherosclerotic plaque instability

AIM：To investigate whether selective inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.METHODS：In in vitro study, casodex (20 μmol/L), rapamycin (10 nmol/L) or mTOR-siRNA (30 nmol/L) was used to treat mouse macrophage cell line RAW 264.7. Inflammation-related cytokines secreted by macrophages were measured by means of ELISA. Ultrastructural changes of the macrophages were examined by transmission electron microscopy. The mRNA and protein expression levels of Akt, mTOR and autophage-related protein Beclin 1 were assayed by real-time fluorescence quantitative RT-PCR and Western blotting. The expression of autophagy-related indicator LC3-II was detected by immunofluorescence and Western blotting. In in vivo study, 24 New Zealand white rabbits underwent balloon-induced abdominal aortic wall injury and were fed with a diet of 1% cholesterol for 8 weeks. The rabbits were randomly divided into control group (n=8), casodex group (1.0 mg·kg-1·d-1, n=8) and rapamycin group (0.5 mg·kg-1·d-1, n=8). Four weeks after drug administration, intravascular ultrasound (IVUS) was carried out to observe the plaque imaging. Ultrastructural changes of the macrophages and the protein expression of Akt, mTOR and LC3-II in the macrophages were also measured.RESULTS：In in vitro study, more typical autophagosomes were detected in casodex-, rapamycin- or mTOR-siRNA-treated cells. The expression level of LC3-II increased, but Beclin 1,p-Akt and p-mTOR significantly decreased in the 3 treatment groups. The concentration of IL-10 decreased while IFN-γ significantly increased in the treatment groups. In in vivo study, IVUS found that external elastic membrane area (EEMA),plaque area(PA) and plaque burden (PB) significantly decreased in casodex and rapamycin treatment groups. Expression of LC3-II increased significantly in the 2 treatment groups. The staining of RAM-11 and p-mTOR in the macrophages was significantly reduced as compared with control group.CONCLUSION：Selective inhibition of PI3K/Akt/mTOR signaling pathway reduces the infiltration of macrophages and stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.     

Expression of tissue factor in cerebral microvascular thrombosis in rats

AIM: To study the expression of tissue factor (TF) in cerebral microvascular thrombosis and its dynamic changes in rats. METHODS: 50 female SD rats were randomized to control group, 2, 4, 6, and 24 hours after thrombosis groups, 10 rats in each group. The model of cerebral microvascular thrombosis was induced by photo-chemical method. ELISA and immunohistochemistry methods were used to observe the changes of TF contents in blood plasma and the expression of TF in cerebral microvascular in each group. RESULTS: Cerebral thrombosis was induced by photo-chemical method successfully. The TF content in plasma was obviously higher in 4 h and 6 h groups than that in control group (P＜0.01). TF intensely expressed on vascular endothelial cells in thrombosis groups and negatively expressed in control group. The average A value of TF in 6 h group and 24 h group was significantly higher than that in 2 h and 4 h groups (P＜0.01). It was also found that vWF content in plasma was obviously higher in 2 h group (P＜0.01) and 4 h group (P＜0.05) group than that in control group. AT activity in plasma showed obviously increased in 24 h group, compared with that in the others (P＜0.05). CONCLUSION: These findings suggest the photochemical infarct model is convenient to investigate cerebral thrombosis. TF plays an important role in thrombosis in this model.     

Expression of connective tissue growth factor in the renal tissue of rats with unilateral ureteral obstruction

AIM: To detect the expression of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in rat unilateral ureteral obstructive (UUO) nephropathy animal model, and to observe the kinetic changes at different stages of firosis. METHODS: Male SD rats were subjected to either left ureteral ligation or sham operation, then killed at 3, 7, 14, 21 or 28 days after UUO or sham operation (n=6 at each time point). HE, Masson or PAS staining were applied to the renal tissue sections. The extent of tubulointerstitial injury was determined by Banff classification. RESULTS: The extent of tubulinterstitial fibrosis became serious with the time of obstruction. Tubules were mostly atropic and replaced by proliferative fibrous tissue at day 28. The expression of CTGF and α-SMA were consistent with the damage of tubulointerstitial. The positive correlation among CTGF or α-SMA and the tubulointerstitial injury scores were significant. The expression of TGF-β1 came to peak at day 7 to 14, and gradually decreased at day 21 and 28. CONCLUSION: These results indicate that the expression of CTGF may be upregulated by TGF-β in UUO rats, and CTGF may be involved in tubulointerstitial fibrosis through the development of myofibroblasts.     

Expression of matrix metalloproteinase and tissue inhibitor of metalloproteinase in lung tissue of hypercapnia rat model

AIM: To study the expression of matrix metalloproteinase-9(MMP-9), matrix metalloproteinase-2(MMP-2) and the tissue inhibitor of metalloproteinase(TIMP-1) in the lung tissue of the hypercapnia rat.METHODS: Forty Wistar rats were randomly divided into a control group (group A, n=20) and hypercapnia group (group B, n=20). Group B received mix gas exposure (6% CO2, 21% O2, 72% N2) 7 h daily for 4 weeks. The parameters we would examine were as follow: arterial blood gas; the mean pulmonary artery pressure;MMP-2,MMP-9, TIMP-1, and NE activity in lung tissue. Masson pigmentation of elasticity fibre was analyzed by computer image analyzer. Histopathological changes of lung tissue were observed under light microscope. The protein expression of MMP (MMP-2, MMP-9) and TIMP (TIMP-1) in lung tissue were determined by immunocytochemistry.RESULTS: Decompensate respiratory acidosis (pH=7.20±0.04, PaCO2=7.84±0.15) developed in group B. The mean pulmonary artery pressure were similar between groups B and A (P>0.05). Tissue edema in the lung, endothelial cell damage of the small blood vessels, pulmonary micro thrombus formations and increased pulmonary capillary permeability were observed in group B. NE activity increased significantly (P<0.01). However, no significant change of MMP-2, MMP-9, TIMP-1 activity was found in group B and group A (P>0.05). There was significant decrease in the relative content of elasticity fibre in lung tissue in group B compared to group A (P<0.01). The expression of MMP-2 protein in the lung tissue of group B was lower than that in group A (P<0.01), but the expression of both MMP-9 and TIMP-1 proteins in the lung tissue in group B were higher than those in group A (P<0.01).CONCLUSION: Hypercapnia rat model is successfully reproduced by exposure of animals to the mix gas exposure (6% CO2, 21% O2, and 72% N2). The pulmonary artery pressure is not affected by hypercapnia. High concentration of CO2 causes increase of NE activity and decrease in the relative content of elasticity fibre. High concentration of CO2 causes the increase of MMP-2 protein expression and decrease in the MMP-9 and TIMP-1 protein expression.     

Significance of soluble CD40 ligand in the vulnerability of coronary atherosclerotic plaque

AIM: To evaluate the significance of serum soluble CD40 ligand (sCD40L) in the vulnerability of coronary atherosclerotic plaque, the relationship between the level of sCD40L and the stenosis degree of the coronary artery by the coronary angiography (CAG), and other inflammatory factors. METHODS: According to WHO diagnostic criterior of coronary heart disease (CHD) and the results of CAG, 84 cases of CHD and 20 cases of non-CHD (NCHD) were included in this study. 84 cases of CHD were divided into three groups: 30 cases in acute myocardial infarction (AMI) group, 30 cases in unstable angina (UA), 24 cases in stable angina (SA). The sera levels of sCD40L in four groups were detected by the enzyme-linked immunosorbent assay (ELISA) and the results were expressed with μg/L. CAG were all conducted in four cases and the results were further evaluated by Jenkins score. ESR and CRP were detected at the same time. RESULTS: The sera levels of sCD40L in four groups were significantly different (P<0.01). The level of sCD40L in AMI group (8.48±4.13) μg/L was higher than that in SA group (4.36±2.68) μg/L, P<0.01 and NCHD group (4.12±1.96) μg/L, P<0.01. The level of sCD40L in UA group (8.72±4.26) μg/L was higher than that in SA group and NCHD group (P<0.01). The level of sCD40L in UA group was slightly higher than that in AMI group, but the difference of two group is not significant (P>0.05). The level of sCD40L in SA group was slightly higher than that in NCHD group, but the difference of two group is not significant (P>0.05). The sera levels of sCD40L in CHD were significantly and positively correlated with Jenkins score (r=0.524, P<0.01). The sera level of sCD40L was positively correlated with the levels of CRP and ESR. CONCLUSION: The sera levels of sCD40L in the patients with various types of CHD are significantly different. The level of sCD40L in the patients with AMI and UA are significantly higher than those in SA and NCHD groups, which may reflect the vulnerability of coronary atherosclerotic plaque. The sera levels of sCD40L is increased with the increasing number of diseased coronary branches and Jenkins score, suggesting that sCD40L promotes atherosclerosis and also can be used as a parameter to predict pathological severity of coronary atherosclerosis. The level of sCD40L is obviously correlated with the levels of CRP and ESR.     

Expression of galectin-3 in human coronary artery and its correlation with plaque structural stability

AIM To investigate the relationship between the expression level of galectin-3 and the stability of plaque structure in human atherosclerotic plaques. METHODS The coronary specimens from autopsy cases （n=84） were collected. Among them, 22 cases had coronary atherosclerotic lesions without sudden death of coronary heart disease （A1 group）, 20 cases were sudden death of coronary heart disease without secondary lesions （A2 group）, 24 cases were sudden death of coronary heart disease with secondary lesions （A3 group）, and 18 cases without heart disease were used as normal control group （control group）. The intimal thickness, necrotic lesion thickness, fibrous cap thickness and the degree of lumen stenosis were measured by routine HE staining in all coronary arteries. The foam cells in the lesion were marked by CD68 and counted. The expression of galectin-3, CD68 and matrix metalloproteinase-2 （MMP-2） in coronary artery intima was detected by immunohistochemical staining, Western blot and RT-qPCR. The correlation between above factors and the structural stability of atherosclerotic plaques was also analyzed. RESULTS Compared with control group, the intima and necrotic lesions were thickened, the fiber cap was thinned, and the degree of lumen stenosis were increased in A1~3 groups （P<0.05）. The number of foam cells in the atherosclerotic focus was increased （P<0.05）. The protein and mRNA levels of galectin-3, CD68 and MMP-2 in the lesions showed an increasing trend from normal group to A1~3 groups （P<0.05）. The expression of galectin-3, CD68 and MMP-2 in atherosclerotic lesions was positively correlated with intimal thickness and necrotic lesion thickness, and negatively correlated with fibrous cap thickness. CONCLUSION The expression of galectin-3 in human coronary atherosclerotic lesions is increased, which is related to the stability of atherosclerotic plaques.     

Effects of herbs of activation blood on atherosclerotic plaque morphology in ApoE gene-deficient mice

AIM: To observe the effects of six common traditional Chinese herbs of activating blood, paeoniae rubra radix, salviae miltiorrhizae radix, ligustici, rhizome, notoginseng radix, pruni persicae semen and wine staemed radix et rhizome, on atherosclerotic plaque structure and stabilization in ApoE gene-deficient mice. METHODS: Four sections of the aortic root were choosen and stained with hematoxylin and masson. All sections were measured with Image-Pro Plus Version 4.5.1 (IPP) software. RESULTS: Compared with the model group, plaque area corrected by cross-sectional vessel wall area reduced significantly in salviae miltirrhizae radix treatment group, lipid core area reduced in paeoniae rubra radix group, pruni persicae semen and wine steamed radix et rhizome treatment group, minimum thickness of fibrous cap became thicker significantly in salviae miltiorrhizae radix, ligustici, rhizome, pruni persicae semen and wine steamed radix et rhizome treatment group. CONCLUSION: These Chinese herbs may stabilize the atherosclerotic plaques in ApoE gene-deficient mice by interfering their structure, but their effects do not parallel with their activating blood efficacy in traditional Chinese medicine.     

Study on the atherosclerotic lesions in coronary artery of apoE gene knockout mice

AIM: To study the distribution and composition of the atherosclerotic lesions in the coronary artery of apoE gene knockout (-/-) mice, and to search for the mechanism of its occurrence and development. METHODS: The successive sections of the hearts of apoE-/- mice were made. All the coronary trunk and branches in myocardium from the opening of coronary artery leaving aorta were traced continuously by Movat staining. The length of the lesions and the distance from the beginning of the coronary was calculated depending on the numbers of sections. The caliber of the vessels was measured with computer and the components of the lesions was also observed by Movat staining. RESULTS: There were two kinds of lesions in apoE-/- mice, extending lesions which directly extended from the beginning of coronary artery and in situ lesions which formed at the branches of the coronary arteries. The in situ lesions tended to locate at the papillary muscles and near the bifurcation of branches in left ventricle. More large lesions in apoE-/- mice of 112 weeks than those of 60 weeks (P＜0.05) were observed. Infiltration of inflammatory cells in the adventitia was usually ahead of the two sorts of atherosclerotic lesions. The component of proteoglycan was decreased and the component of lipid was increased with the enlargement of the in situ lesions. Some large lesions may obstruct the small vessels. CONCLUSIONS: There are two kinds of atherosclerotic lesions (extending lesion and in situ lesion) within the coronary artery of apoE-/- mice. Both kinds of lesions are gradually increased with age in size. The infiltration of inflammatory cells in adventitia and the hemodynamic oppression by myocardial contraction are related to the occurrence and development of these lesions.     

Effects of pituitary adenylate cyclase-activating polypeptide on cardiac remodeling in coronary atherosclerotic rabbits

AIM:To study the intervention effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cardiac remodeling during the development of rabbit coronary atherosclerosis. METHODS:60 male New Zealand rabbits were equally divided into 3 groups randomly: control group (C group), atherosclerosis model group (A group) and PACAP intervention group (P group). At the 4th, 8th and 12th week, 5-6 cases of rabbits in each group were sacrificed, cardiac tissue with coronary arteries were harvested to make paraffin sections. The sections were stained with hematoxylin-eosin and van Gieson separately. The qualitative observation and/or quantitative analysis were made by light microscope. RESULTS:（1）There was no lesion in C group. For A group and P group, there were plaques in large epicardial coronary arteries and small coronary arteries; an impressive accumulation of collagen was also observed in myocardium. In P group, the lesions of small coronary arteries were less serious, and the degrees of perivascular and myocardial fibrosis also appeared to be less.（2）For A group, the wall-to-lumen ratios in small coronary arteries were significantly greater at the 12th week (2.58±1.54) than C group (1.34±0.58) and P group (1.39±0.48) （P<0.05）； and the width of cardiomyocyte (13.85 μm±2.27 μm) was already remarkably narrower than C group (14.68 μm±2.40 μm) at the 8th week (P<0.05) and narrower significantly than C group and P group at the 12th week. （3）There were not difference significantly between the above-related parameters of P group and C group (P>0.05). CONCLUSION:Structure changes exist in coronary arteries and myocardium during the development of rabbit coronary atherosclerosis, PACAP can inhibit the cardiac remodeling.     

Expression and modulation of connective tissue growth factor in renal interstitial fibrosis

CTGF, a member of the CCN family of immediate early genes, is a recently discovered profibrotic growth factor, which is involved in many pathophysiologic procedures. CTGF acts as a downstream effector of TGF-β acting on interstitial cells to enhance the progression of fibrotic renal diseases. It has been shown that CTGF gene expression can be induced or blocked by some kinds of cytokine and drugs. It is an interesting candidate target for future intervention strategies of renal interstitial fibrosis.     

Effects of Danggui Buxue decoction on endothelial progenitor cells,serum VEGF and SDF-1 in atherosclerotic rabbits

AIM: To investigate the effects of Danggui Buxue decoction (DBD) on serum vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1), as well as the activity of circulating endothelial progenitor cells (EPCs) in atherosclerotic rabbits. METHODS: The model of atherosclerosis was established using immune injury and fatty diet for 4 weeks in New Zealand rabbits (n=25). All modeled rabbits were randomized into 5 groups with 5 animals in each group. The rabbits in atherosclerosis group were intragastrically administered with distilled water. The rabbits in simvastatin group were treated with simvastatin at the dose of 1.7 mg/kg. The rabbits in DBD high-dose, middle-dose and low-dose treatment groups were given DBD at the doses of 6 g/kg, 3 g/kg and 1.5 g/kg, respectively. All drugs were given once a day for 2 weeks. After treatment, the levels of serum VEGF and SDF-1 were measured. The mononuclear cells isolated from the rabbit peripheral blood were cultured for 7 days in vitro, and then attached cells were cultured with both DiI-ac-LDL and FITC-UEA-1 for identification. The proliferation was detected by MTT method. The cell migration was observed using Transwell chambers. The adhesion determination and in vitro angiogenesis assay were also performed. RESULTS: Compared with atherosclerosis group, the levels of serum VEGF and SDF-1 were elevated (P<0.05), the proliferation, migration, adhesion and angiogenesis of EPCs were all improved in DBD high-dose, middle-dose treatment groups and simvastatin group (P<0.05). CONCLUSION: DBD elevates the levels of serum VEGF and SDF-1 to improve the activity of EPCs in the process of atherosclerosis.     

Effects of rapamycin-treated HSP60-pulsed dendritic cells on th e progression of the atherosclerotic plaque in mice

AIM:To evaluate whether tolerogenic dendri tic cells (DC) loaded with heat shock protein 60 (HSP60) inhibit the progression of aortic atherosclerotic plaque in hypercholesterolemic apolipoprotein E (Apo- E) -null mice.METHODS:Bone marrow derived DC of the mice were loaded with HSP 60 and co-cultured with rapamycin to generate tolerogenic DC.The tolerogenic DC ,DC loaded only HSP60 and PBS were injected into the ApoE-null mice at 8 weeks of age for three times at a one-week interval.8 weeks after the last injection,aorta were harvested for HE staining and anti-CD4+T cell immunostaining.Resp onses of pleenic cells to HSP60 were also evaluated.RESULTS:Compared with DC,DCHSP60 expressed higher levels of CD86,and stimulated T lymphocytes to proliferation significantly,while the tolerogenic DC expressed lower levels of CD86,and inhibited T lymphocytes to p roliferation.After immunization with different injection,the numbers of CD4+ T cells in plaque were increased significantly in DCHSP60 group vs in PBS g roup (P<0.01).On the other hand,they were reduced significantly in rap-DC HSP60 group vs in PBS group (P<0.01).Plaque areas in the tolerog enic DC group were smaller than that in the PBS group (P<0.01).Stimulated by HSP60,pleenic cells in tolerogenic DC group secreted more IL-10,while in DC HSP60 group more IFN-γ secretion was observed.CONCLUSION:HSP60 specific tolerogenic DC immunization attenuate d the progression of plaque,indicating a new immune therapy for atherosclerosis.     

Effects of adventitial inflammation of coronary artery on the formation of atherosclerotic lesions

AIM: To study the pathogenesis of atherosclerotic lesions which were closely related to the adventitial inflammation of coronary artery (CA) in apolipoprotein E gene knockout (apoE-/-) mice. METHODS: The hearts of apoE-/- mice were cut consecutively. Three kinds of CA samples (① Infiltration of inflammatory cells at CA adventitia, without lesion; ② Infiltration of inflammatory cells at CA adventitia, with the top of extending lesion directly from aorta; ③ Infiltration of inflammatory cells at CA adventitia, with mature lesion) were chosen to represent the three stages of atherosclerotic lesion formation. HE staining, Movat staining, immunohistochemical staining and electron transmission microscopy were used respectively to identify the types of the inflammatory cells infiltrated at adventitia of coronary artery. RESULTS: The constituent ratio of macrophages which infiltrated in the CA adventitia without atherosclerotic lesions, of neutrophils which were involved in the CA adventitia with young atherosclerotic lesions and of lymphocytes in the CA adventitia with mature lesions, were 60.00%, 57.65% and 66.67%, which were higher than those in the other two groups, respectively (P<0.01). CONCLUSION: It was showed that CA adventitial inflammation might be an early event inducing the formation of atherosclerotic lesions. The CA adventitia undergoes a process from acute to chronic inflammation during the formation of atherosclerotic lesions.     

Effects of pravastatin combined with warfarin on steroid-induced femoral head necrosis in rabbits

AIM: To investigate the role of pravastatin combined with warfarin in treating steroid-induced osteonecrosis of femoral head (SIONF) in rabbits. METHODS: Forty-eight Japan healthy adult female rabbits were randomly divided into 3 groups: control group (CTR group, 12 rabbits), experimental group (SIONF group, 18 rabbits) and pravastatin+warfarin treatment group (PW group, 18 rabbits). The rabbits in SIONF group and PW group were injected with methylprednisolone (20 mg/kg) intramuscularly to establish the animal model of SIONF. The rabbits in CTR group were only injected with normal saline intramuscularly. The rabbits in PW group were also received pravastatin (2.5 mg·kg^-1·d^-1) and warfarin(1.5 mg·kg^-1·d^-1)orally. The blood samples were collected from 2 rabbits in each group at time point of pre-experiment and 2, 4, 6, 8, 10, 12 weeks post-experiment for measuring the contents of total cholesterol (TC) and triglyceride (TG), prothrombin time (PT) and activated partial thromboplastin time (APTT). In the latter 4 time points, the X-ray examination of bilateral hip joints were taken, and the animals were then killed to collect the bone tissues for ultrastructural observation under transmission electron microscope. The SIONF incidence rate was also determined. RESULTS: From the 6th week to the end of experiment, the level of TC in PW group was higher than that in CTR group (P<0.05), but lower than that in SIONF group (P<0.05). The level of TC in SIONF group was higher than that in CTR group (P<0.01). The levels of TG showed the similar changes as TC. From the 2nd week of experiment, significant prolonged PT was observed in PW group as compared with CTR group and SIONF group (P<0.01). On the contrary, significant shortened PT in SIONF group was found (P<0.01). The changes of APTT were similar to the changes of PT. Compared with SIONF group, the bone tissues in PW group showed significant decrease in the conversion rate of intramedullary fat in femoral head and the rate of bone recess. No thrombus formation was observed, and bone karyopyknosis and autolysis were also significantly reduced. In PW group, the SIONF incidence rate was 31 % (5/16), significantly lower than 63% (10/16) in SIONF group (P<0.05). CONCLUSION: Combination of pravastatin with warfarin reduces the incidence rate of SIONF in rabbits, indicating a relation with the improvement of hyperlipid and hypercoagulant state.     

Forestry expansion and land-use patterns in the Nelson Region,New Zealand

The expansion of plantation forestry in New Zealandduring the last century has altered the landscape and will continue to do so in the future. The implementation of recent resource management policy, the 1991 Resource Management Act (RMA), will also influence the impact of future plantation expansion on the landscape. It is necessary to analyze current land-use patterns in order to predict effects on future landscapes. This study analyzed the current land-use patterns of the Nelson region by examining the relationship between land-use and site conditions and by characterizing the distribution pattern of land-use depending on distance from the city. The distribution pattern was considered from an economic perspective, based on the Barlowe’s model of land use distribution. The relationship between the current land-use pattern and previous land management policy was also examined using the Land Use Capability. Consequently, in addition to the physical attributes of the land, the influence of land-use management policy was obvious. Thus the RMA will undoubtedly influence land-use changes in the future. It is therefore necessary to understand the factors determining changes in land-use patterns in conjunction with the district plan of the RMA to predict future changes in land-use.This revised version was published online in May 2005 with corrections to the Cover Date.