中性粒细胞明胶酶相关脂质运载蛋白(NGAL)信号通路在猪静脉桥血管早期再狭窄模型中的表达及作用

目的研究中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin,NGAL)信号通路在猪静脉桥早期再狭窄模型中的表达及其作用机制,从而探索NGAL信号通路在冠状动脉旁路移植术术后静脉桥血管早期再狭窄发生中的作用及相关机制。方法选择健康的普通长白猪18只,体重25~30 kg。对猪静脉桥再狭窄模型建立前、建立后7 d、14 d、30 d共4个时间点的静脉桥标本进行检测,并与未移植的大隐静脉进行对比,采用苏木素-伊红(HE)染色,Masson染色,免疫组织化学等方法,观察静脉桥血管内膜增生、平滑肌细胞迁移和血管重塑的情况,并对NGAL、基质金属蛋白酶(MMP)9、MMP2、组织金属蛋白酶抑制剂(TIMP)1等因子在不同桥血管中的表达变化情况进行检测。结果通过HE与Masson染色,随着建模时间的推移,桥血管胶原基质逐渐降解,肌纤维增厚并向内膜迁移,血管重塑,导致桥血管的狭窄。通过免疫组织化学结果可以看出,动物模型中NGAL、MMP9、MMP2因子在正常静脉桥血管中极少表达甚至不表达。建模后第14 d血管中膜层开始出现NGAL表达,第30 d中膜层NGAL表达达到高峰,并且内膜层开始出现表达。MMP9、MMP2在建模后第7 d开始出现表达,表达高峰出现在建模第14 d,30 d后表达趋于正常水平。正常血管壁TIMP1表达较少,建模后第14 d血管中的表达达到高峰。结论 NGAL、MMP9、MMP2、TIMP1等因子可能参与了桥血管早期再狭窄的形成,NGAL作为起始因子,导致MMP9、MMP2的表达增加,参与基质的降解和平滑肌细胞向内膜迁移。TIMP1等作为负性因子,对维持自身平衡可能起到重要的作用。     

Ⅳ型胶原蛋白和基质金属蛋白酶2在移植静脉中表达的研究

目的观察大鼠自体静脉移植后内膜增生 (IH)平滑肌细胞 (SMC)增殖、Ⅳ型胶原和基质金属蛋白酶 2 (MMP 2 )的变化。方法建立大鼠自体静脉移植模型 ,于术后不同时间处死动物。标本行HE、Verhoeff染色、SP法五溴脱氧脲嘧啶 (5′ Brdu)、Ⅳ型胶原、MMP 2免疫组化染色 ,原位杂交检测Ⅳ型胶原和MMP 2mRNA的表达。计算机图像分析仪测量移植静脉内膜 /中膜厚度、面积比 ,阳性细胞百分比 ,Ⅳ型胶原和MMP 2蛋白和mRNA阳性表达百分比。结果术后 1周移植静脉开始形成新内膜 ,2～ 4周内膜增生明显 ,8～ 12周 ,内膜增生有下降趋势。 5′ Brdu阳性细胞百分比变化趋势与内膜增生趋势相似。与正常静脉比较Ⅳ型胶原阳性区域于术后 1周明显减少 (P <0 0 5 ) ,2～ 8周管壁未见阳性区域。MMP 2在术后 1周表达明显增加 ,2周达高峰 ,8～ 12周恢复至术前水平。Ⅳ型胶原mRNA术后 3d一过性表达增多 (P <0 0 5 )。MMP 2mRNA术后 3d即迅速增高 ,1周达高峰 (P <0 0 5 )。结论静脉移植术后MMP 2高表达导致局部Ⅳ型胶原降解和破坏 ,是SMC向内膜迁移、增殖的重要原因。     

基质金属蛋白酶及其抑制剂与血管再狭窄

基质金属蛋白酶（matrix metalloproteinase,MMP）是由锌和钙依赖性肽链内切酶多基因家族组成,能消化降解细胞外基质（extra cellular matrix,ECM）的几乎所有成分,是参与ECM降解的最主要的酶。血管重建术后,血管损伤、血流动力学改变、炎症、血栓形成及纤溶激活、氧化应激等因素都可促进MMP的表达和激活,活化的MMP降解ECM,促进血管平滑肌细胞（vascular smooth muscle cell,VSMC）迁移增殖,导致新内膜增生和血管重塑,最终导致血管再狭窄。     

重组arresten蛋白对自体移植静脉内膜增生的抑制作用

目的 观察原核表达人arresten重组蛋白对自体移植静脉内膜增生的抑制作用.方法 用pRSET原核表达系统表达并纯化人arresten重组蛋白.将Wistar大鼠颈外静脉移植于腹主动脉,建立大鼠自体静脉移植模型,实验分3组:假手术组、移植对照组和移植实验组.自术后第3天起,皮下注射给予arresten重组蛋白(每日4 mg/kg体重)处理.4周后取移植静脉组织标本,进行病理组织学观察与免疫组织化学染色分析.结果 移植组移植静脉均呈现典型的内膜增生、肥厚,导致血管管腔狭窄;新生内膜主要有过度增殖的α-SMA染色阳性平滑肌细胞组成.移植实验组移植静脉内膜增生受到明显抑制,新生内膜面积(0.12±0.07)mm2及新生内膜/中膜面积比(0.373±0.085)均显著低于对照组[(0.38±0.11)mm2,1.621±0.086,P<0.01];并且实验组移植静脉新生内膜细胞PCNA标记指数显著低于对照组[(15.62±3.97)%比(56.36±3.49)%,P<0.01].结论 重组arresten蛋白通过抑制新生内膜平滑肌细胞的增殖能有效抑制自体移植静脉内膜增生的发生发展,在防治血管重建术后再狭窄方面显示出良好的应用前景.     

血小板源性生长因子及其受体与动脉粥样硬化

1 血小板源性生长因子(platelet-derived growth factor,PDGF)概述 动脉粥样硬化(atherosclerosis,AS)及其并发症是目前全球人口死亡的首要原因.动脉中膜平滑肌细胞(smooth muscle cell,SMC)增殖是AS病变的病理基础,来自内膜层SMC的巨噬细胞在内皮下蓄积,使得斑块形成、破裂,最终导致血管闭塞并发生临床事件[1]. 30年前,研究人员从血小板中发现了一种可刺激SMC生长的血清衍生因子,即为血小板源性生长因子(platelet-derived growth factor,PDGF).PDGF在损伤部位蓄积,并在细胞外基质增殖使损伤扩大,它和血小板源性生长因子受体(platelet-derived growth factor receptor,PDGFR)一起组成血小板源性生长因子家族.     

终末期肾病血液透析患者血浆COMP表达与动静脉内瘘失功及预后的相关性研究

目的 检测终末期肾病(end-stage renal disease,ESRD)血液透析患者血浆软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)表达,并分析COMP与动静脉内瘘(arteriovenous fistula,AVF)失功及预后的相关性.方法 选取2015...     

碱性成纤维细胞生长因子真核表达载体的构建及表达

目的构建人碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)真核表达质粒,研究其在体内外的表达.方法通过基因克隆技术,构建并大量制备pcDNA3.1/myc-His(-)-bFGF真核表达体系,RT-PCR和细胞免疫组织化学方法检测体外瞬时表达情况;直流电脉冲介导重组质粒pcDNA3.1/myc-His(-)-bFGF和pCD2-血管内皮细胞生长因子121(vascular endothelial growth factor,VEGF121)在兔颈部肌肉瓣内转移并表达,测定其促血管生成的生物学效应.结果构建的pcDNA3.1/myc-His(-)-bFGF真核表达体系成功转染体外培养HeLa细胞,目的基因在mRNA水平和蛋白水平均有表达.pcDNA3.1/myc-His(-)-bFGF和pCD2-VEGF121重组质粒分别转染在体肌瓣,获得外源基因高水平表达.转基因肌肉发生血管增生、血流增强的生物学效应.结论构建了人bFGF真核表达质粒,并可在体内外顺利表达,为进一步组织移植或组织工程基因治疗研究奠定了基础.     

血管内皮生长因子调节小鼠系膜细胞基质金属蛋白酶及其组织抑制物的表达

目的：研究血管内皮生长因子（VEGF）对体外培养的小鼠系膜细胞表达基质金属蛋白酶（MMPs)和金属蛋白酶组织抑制物（TIMPs)的调节作用。方法：对体外培养的小鼠系膜细胞应用人重组VEGF孵育12h，应用蛋白质印迹法检测细胞培养液MMP2、MMP9、TIMP1、TIMP2的蛋白质水平；应用白明胶酶谱法检测MMP2、MMP9的活性；应用RT－PCR检测系膜细胞TIMP1、TIMP2 mRNA表达。结果：VEGF（10ng/ml)可提高小鼠系膜细胞MMP2和MMP9蛋白质释放（和对照组相比分别提高43％和34％，P＜0．05），MMP2和MMP9活性分别提高17％和26％（P＜0．05）。同时，VEGF可呈剂量依赖地下调小鼠系膜细胞TIMP1和TIMP2蛋白质和mRNA的表达，VEGF（25ng/ml)可减少小鼠系膜细胞TIMP1和TIMP2蛋白质释放分别为43％和67％（P＜0．05）；并抑制TIMP1和TIMP2 mRNA的表达（分别下降41％和59％，P＜0．01）。结论：VEGF使系膜细胞MMPs蛋白质表达增加、活性增强，同时下调TIMPs mRNA和蛋白质的表达，可能在增生性肾小球肾炎的发病机制中起一定的作用。     

重组血管生成抑制因子-1对增生性瘢痕组织血管及其相关因子的影响

目的 研究基因转染血管生成抑制对兔耳增生性瘢痕组织血管及其相关因子表达的影响.方法 将基因重组血管抑制剂Ad-METH-1作用于兔耳增生性瘢痕,用微循环显微镜检、组织学染色、免疫组织化学染色等方法,研究Ad-METH-1对兔耳瘢痕组织增生、血管生成及血管内皮细胞生长因子(vascular endothelial cell growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)表达的影响,探讨基因转染血管生成抑制对增生性瘢痕的影响.结果 Ad-METH-1注射后30 d,实验组瘢痕组织微血管计数为12.38±2.56,VEGF阳性细胞百分比为17.64%,bFGF阳性细胞为18.24%;对照组微血管计数为48.12±6.46,VEGF阳性细胞百分比为31.34%,bFGF阳性细胞为28.26%.结果 显示,实验组瘢痕组织微血管计数低于对照组,两组间差异有统计学意义(P＜0.01);实验组瘢痕组织VEGF及bFGF的阳性细胞百分比均低于对照组,两组间差异有统计学意义(P＜0.05).结论 Ad-METH-1对兔耳瘢痕组织增生、血管生成及VEGF、bFGF表达产生了明确的抑制作用,早期行血管抑制治疗可抑制增生性瘢痕的形成.基因转染血管抑制治疗有望成为一种有效的增生性瘢痕防治方法.     

明胶酶A及其抑制剂、碱性成纤维细胞生长因子及受体在肺鳞癌的表达

目的 探讨明胶酶A(MMP2)及其抑制剂组织金属蛋白酶抑制物(TIMPs),碱性成纤维细胞生长因子(bFGF)及其受体(FGFR)-1在人肺鳞癌(SCCL)中的表达与肿瘤转移及患者预后的相关性。方法采用免疫组织化学链霉抗生物素蛋白-过氧化物酶(SP)法检测30例有转移的肺鳞癌、20例无转移的肺鳞癌、15例正常支气管上皮、12例增生上皮和10例不典型增生上皮中MMP2、TIMP2和bFGF、FGFR-1蛋白的表达。结果 正常支气管上皮→增生上皮→不典型增生→无转移鳞癌→有转移鳞癌的演进过程中,MMP2、TIMP2和bFGF、FGF-1蛋白阳性表达率均逐步显著增高(P<0.05)。有转移癌与无转移癌相比,MMP2、TIMP2和bFGF表达的差异均有显著性(P<0.05),增生 不典型增生上皮与正常上皮相比,MMP2表达的差异具有显著性(P<0.05)。4种蛋白的表达率均与鳞癌TNM分期正相关(P<0.05)。Kaplan meier分析发现MMP2和TIMP2阳性表达患者的术后生存率显著低于阴性表达者(P<0.05)。结论 MMP2、TIMP2和bFGF、FGFR-1过度表达导致肿瘤的浸润转移及预后不良。     

The cyclolignan picropodophyllin attenuates intimal hyperplasia after rat carotid balloon injury by blocking insulin-like growth factor-1 receptor signaling

OBJECTIVE: Smooth muscle cell proliferation (SMC) is a pivotal factor in the development of intimal hyperplasia after vascular injury. A number of growth factors, including insulin-like growth factor-1 (IGF-1), have been shown to be involved in SMC proliferation. We evaluated the effect of picropodophyllin (PPP), a new IGF-1 receptor inhibitor, in the prevention of SMC proliferation and development of intimal hyperplasia after vascular injury. METHODS: The effects of systemic administration of PPP on intimal hyperplasia were studied in a balloon rat carotid injury model. Lesions were quantified by morphometry and SMC proliferation and apoptosis was studied by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and activated caspase 3, respectively. The effect of PPP on rat aortic SMC proliferation and apoptosis was studied in vitro by using cell counting, 3[H]-thymidine incorporation, and a flow cytometry assay for annexin V. Phosphorylation of the IGF-1 receptor, protein kinase B (Akt), and extracellular signal-regulated kinase 1/2 (ERK1/2) in vitro and in vivo were analyzed by using Western blotting. RESULTS: PPP inhibited IGF-1-mediated SMC proliferation in vitro but no significant increase in apoptosis was detected. In rats treated with PPP, a more than a twofold reduction in carotid intima area was observed 2 weeks after balloon injury, a significant decrease in PCNA staining was demonstrated in early lesions, but activated caspase 3 was not detected. In addition, PPP attenuated phosphorylation of the IGF-1 receptor, Akt, and ERK1/2 in IGF-1-stimulated SMCs in vitro, and a reduced phosphorylation of the IGF-1 receptor and Akt was found in balloon-injured carotid arteries in rats treated with PPP. CONCLUSION: These results show that PPP potently blocks IGF-1-mediated phosphorylation of the IGF-1 receptor in SMCs, decreases downstream Akt and ERK1/2 activation, inhibits SMC replication, and subsequently attenuates intimal hyperplasia after balloon injury of rat carotid arteries.     

Smooth muscle cells cultured from human saphenous vein exhibit increased proliferation, invasion, and mitogen-activated protein kinase activation in vitro compared with paired internal mammary artery cells

OBJECTIVE: Smooth muscle cell (SMC) proliferation, invasion, and matrix metalloproteinase (MMP) secretion are key events in the development of intimal hyperplasia, the lesion that causes coronary artery bypass graft (CABG) failure. Saphenous vein (SV) grafts are the most commonly used bypass conduits but are markedly more susceptible to intimal hyperplasia than internal mammary artery (IMA) grafts. We hypothesized that this may be due to inherent functional differences between SV-SMCs and IMA-SMCs. In this study we used paired cultures of SV-SMCs and IMA-SMCs from the same patients and compared their rates of proliferation, invasion, migration, and MMP-2 and MMP-9 secretion. METHODS: SMCs were cultured from explants of paired SV and IMA from 22 patients undergoing CABG. SMC populations of equivalent passage were used to determine proliferation in response to 10% fetal calf serum (FCS), 10 ng/mL platelet-derived growth factor (PDGF), and 10 ng/mL basic fibroblast growth factor (bFGF) by counting cells during a 7-day period. Immunoblotting was used to quantify phosphorylation of p44/42-mitogen-activated protein kinase (MAPK). Invasion and migration rates of paired SMCs were quantified using a modified Boyden chamber technique in the presence or absence of a Matrigel basement membrane barrier (BD Biosciences, Oxford, UK). Conditioned media from invasion assays were analyzed for secretion of MMP-2 and MMP-9 by gelatin zymography. RESULTS: Analysis of areas under curves for 7-day proliferation assays revealed that the number of SV-SMCs in response to FCS, PDGF, and bFGF was 2.1, 2.0, and 2.3 times higher, respectively, than that of paired IMA-SMCs. Basal MAPK activation in SV-SMCs was approximately double that of paired IMA-SMCs. SV-SMCs exhibited a 2.1-fold increase in invasion rate (Matrigel barrier) compared with IMA-SMCs, but migration rates (no Matrigel barrier) and MMP-2 and MMP-9 secretion were similar for the two cell types. CONCLUSIONS: Human SV-SMCs are inherently more proliferative and invasive than paired IMA-SMCs, likely due to a relative increase in p44/42-MAPK activation. These inherent functional differences between SMC of different origins may contribute to the increased prevalence of intimal hyperplasia in SV grafts compared with IMA grafts.     

Effect of galectin-3 in the pathogenesis of arteriovenous fistula stenosis formation

ObjectiveThis study sought to investigate the effect of local expression of galectin-3 in the development of stenotic arteriovenous fistula (AVF).MethodsWe collected stenotic venous tissues, adjacent nonstenotic venous tissues, and blood samples from end-stage renal disease (ESRD) patients with AVF stenosis, while normal venous tissues and blood samples were collected from ESRD patients before AVF creation as controls. Also blood samples were collected from ESRD patients with nonstenosis functional AVF. Galectin-3, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase-9 (MMP-9), and α-SMA expression in the venous tissues were examined by immunohistochemistry, and the ERK1/2 pathway activity in the intima was accessed by western blot. Serum galectin-3 level was measured by ELISA. Thereafter, human pulmonary arterial smooth muscle cells (HPASMCs) were cultured in vitro, and the interaction between Galectin-3 and ERK1/2 pathway in HPASMCs was estimated by western blot.ResultsESRD patients with stenotic AVF had a significant higher serum galectin-3 level than normal controls, and patients with non-stenotic functional AVF. The expression levels of galectin-3, phosphorylated ERK1/2, PCNA, MMP-9, and α-SMA in the stenotic venous tissues were higher than that in the normal venous tissues or the adjacent nonstenotic AVF venous tissues. Correlation analysis showed that the expression of galectin-3 of the neointima was positively correlated with PCNA and α-SMA in the stenotic AVF venous tissues. In HPASMCs, galectin-3 can increase the activity of phosphorylated ERK1/2 and promote the expression of α-SMA.ConclusionIn the stenotic AVF of ESRD patients, expression of the galectin-3 was significantly increased, showing a positive relation with neointima development.     

Stem cell factor and c-kit are expressed by and may affect vascular SMCs through an autocrine pathway

INTRODUCTION: Stem cell factor (SCF) is a membrane-bound and soluble growth factor that activates the c-kit tyrosine kinase receptor. Given the similarities between c-kit and platelet-derived growth factor (PDGF) receptors, we hypothesized that similar to PDGF, SCF/c-kit signaling may play a role in smooth muscle cell (SMC) function and thus the development of intimal hyperplasia. MATERIALS AND METHODS: Human saphenous vein SMCs were harvested from veins procured at the time of bypass grafting. Carotid arteries from rats that were balloon injured (n = 12) at variable time points were compared to sham-operated controls (n = 3). Expression of SCF and c-kit was measured by immunohistochemistry (IHC) and Western blotting. RESULTS: Western blotting revealed that human SMCs express membrane-bound SCF. In separate experiments, we found that this growth factor undergoes proteolytic cleavage to its soluble form following exposure to matrix metalloproteinase-9 (MMP-9), a ubiquitous MMP released at the time of arterial injury. We next evaluated in human SMCs, expression of the SCF receptor, c-kit. Western blotting of human SMC lysates revealed minor but consistent expression of c-kit. IHC demonstrated c-kit expression to be localized to the media. To determine if c-kit is up-regulated during the development of intimal hyperplasia, we evaluated expression of this receptor in a rat carotid balloon injury model. Quantification of IHC staining on injured vessels revealed that c-kit expression within the media was significantly increased at 3, 7, 14, and 28 days following injury (28.1, 30.8, 16, and 10.4% increase over sham controls, respectively, P < 0.05). Furthermore, c-kit expression was prominent within the neointima and maximal at 7 days (53.4 +/- 7.8% of area c-kit positive). CONCLUSION: Human vascular SMCs express the growth factor SCF and its receptor, c-kit. SCF is released from its membrane-bound form via MMP-9. This finding and the dramatic increase in c-kit expression observed in the rat carotid artery after balloon injury suggests SCF/c-kit signaling may affect SMC function via an autocrine pathway.     

Cytotoxic Effects of Basic FGF and Heparin Binding EGF Conjugated with Cytotoxin Saporin on Vascular Cell Cultures

Vascular smooth muscle cell (SMC) proliferation is an integral component of intimal lesion formation. In this study we compared the mitogenic effects of basic fibroblast growth factor (bFGF) and heparin binding epidermal growth factor (HBEGF) and the cytotoxic effects of bFGF and HBEGF conjugated with plant cytotoxin saporin (SAP) on vascular cell cultures. Human vascular SMCs and endothelial cells were cultured and FGF receptor-1 (FGFR-1) and EGF receptor (EGFR) expression were detected by immunohistochemical staining. Cells were grown in 24-well plates. Variable amounts of testing drugs (bFGF, HBEGF, SAP, bFGF-SAP, or HBEGF-SAP) were added to quadruplicate wells after 24 h. Cells without drugs were used as control. The total number of cells was counted at 72 h using a hemocytometer. The cultured human vascular SMCs and endothelial cells expressed both FGFR-1 and EGFR with predominant perinuclear localization. bFGF and HBEGF demonstrated equally potent mitogenic effects on SMC proliferation. SAP alone showed a limited cytotoxic effect on both SMCs and endothelial cells. bFGF had a more potent effect on endothelial cell proliferation than HBEGF. bFGF-SAP was equally cytotoxic for both SMCs and endothelial cells, while HBEGF-SAP had a more selectively cytotoxic effect on SMCs than on endothelial cells. These data suggest that the mitogenic effects of bFGF and HBEGF and the cytotoxic effects of bFGF-SAP and HBEGF-SAP may both be mediated by their corresponding growth factor receptors. Because of its selective cytotoxic effect on SMCs, HBEGF-SAP may become a more attractive agent for controlling intimal lesion formation.     

Production and inhibition of the gelatinolytic matrix metalloproteinases in a human model of vein graft stenosis.

OBJECTIVES: human vein graft stenoses are caused by intimal hyperplasia, a process which is characterised by extensive degradation and accumulation of extracellular matrix. This study investigated the role of the matrix metalloproteinases (MMPs) - the principal physiological mediators of extracellular matrix degradation - in the development of intimal hyperplasia in cultured human long saphenous vein. DESIGN: experimental study. MATERIALS AND METHODS: paired venous segments with the endothelium intact or denuded were cultured in standard conditions for 14 days. At the termination of culture, MMPs were extracted from one half of the tissue, whilst the remainder of the vein was prepared for histological examination. RESULTS: stereologic analysis revealed that the endothelium intact veins developed a significantly thicker neointima when compared to the denuded venous segments (20 micron v. 0 micron, p=0.006). Quantification of MMPs by substrate gel enzymography demonstrated that the development of a neointima was associated with increased production of the gelatinolytic MMP-9 (p=0. 03) in intact veins. Immunocytochemistry showed that the MMP-9 localised to the internal elastic lumina, which suggested a role in facilitating smooth-muscle-cell migration into the intima. The role of MMPs-2 and -9 in intimal hyperplasia was further investigated by culturing intact venous segments with a therapeutic concentration of doxycycline--a potent MMP inhibitor. These experiments demonstrated that a therapeutic dose of doxycycline significantly reduced neointimal thickness (control 21 micron, doxycycline 10 mg/l-5.5 micron), in conjunction with a significant reduction in the production of MMP-9. CONCLUSIONS: these data suggest that elevated levels of MMPs may play a significant role in the development of human intimal hyperplasia and that inhibition of these enzymes may offer a potential therapeutic strategy for the prevention of hyperplastic lesions.     

Experimental study of chitosan inhibiting vascular intimal hyperplasia of rabbit arteriovenous fistula

Objective To investigate the effete of chitosan on rabbit carotid artery internal jugular vein fistula intimal hyperplasia and its regulation on TLR4/NF-κB signaling. Methods A total of 28 New Zealand white rabbits were randomly divided into the control group(n=4), the model group(n=12) and the chitosan group(n=12). Model group and chitosan group rabbits were established respectively carotid artery internal jugular vein fistula models. After AVF surgery, chitosan was smeared on venous blood vessels and anastomosis. After 4, 6 and 8 weeks, the rabbits were separately sacrificed and the AVF venous vascular tissues were taken. The pathological changes of AVF venous vascular tissue in each group were observed. The changes of α - SMA were detected by immunohistochemistry method. The mRNA expressions of PCNA and TLR4 in the tissues were measured by Real-time PCR. At the same time, the protein expressions of PCNA, TLR4, MyD88 and NF-κB were detected by Western blotting. The experimental data were processed by two-factor analysis of variance in statistics. Results (1) After 4 weeks, vascular intimal was thicked in mdel group. In intimal hyperplasia, α-SMA was staining, and then proliferation of vascular smooth muscle cell was significant. As time increasing, more intimal hyperplasia shown obviously , the expression of α-SMA significantly increased. Compared with model group, chitosan group significantly reduced the degree of intimal hyperplasia, the level of α - SMA was significantly decreased,vascular smooth muscle cell proliferation was also extraordinarily decreased. (2) Compared with control group, the expression levels of PCNA, TLR4, MyD88 and NF-κB increased with time. The indices of Chitosan group were markedly higher than control group, but significantly lower than model groups. Conclusion Chitosan can inhibit the proliferation of rabbit VSMCs. The mechanism may be concerned in down regulating TLR4- mediated signaling pathway, reducing the possibility of intimal hyperplasia of rabbit AVF venous blood vessels.     

Pre-existing histopathological changes in the cephalic vein of renal failure patients before arterio-venous fistula (AVF) construction.

BACKGROUND: Native cephalic vein remains the superior dialysis conduit, even 30 years after it was first described. However, up to 37% of hemodialysis patients develop progressive stenosis in the venous circuit of arterio-venous fistula (AVF), which may later cause thrombosis and occlusion. MATERIAL AND METHODS: To study the pre-existing morphological changes in the wall of the cephalic vein before AVF construction, we collected 23 cephalic vein specimens from 3 normal, young trauma patients and 20 renal failure patients. The samples were collected at the time of vascular repair in the first group and AVF construction in the second group. Sections were prepared and stained with hematoxylin & eosin (H&E), Masson's trichrome and Verhoff von Gieson's stains. RESULTS: Compared with normal cephalic veins, all pre-access cephalic veins showed generalized thickening of the wall due to intimal hyperplasia and replacement by collagenous, fibrous tissue. Other changes were disruption or loss of internal elastic lamina in 9 (45%) patients, loss of endothelial cell layer in 6 (30%), atrophy or loss of the muscle layer in 6 (30%), mucoid or myxoid degeneration in 6 (30%), inflammatory cell infiltration of the wall in 5 (25%), mural calcification in 3 (15%) and telangiectasia in 2 (10%). Another important finding was the marked accumulation of spindle-shaped smooth muscle cells (SMCs) on the de-epithelialized intimal surface in areas of intimal hyperplasia. CONCLUSION: In conclusion, most of the apparently normal cephalic veins of the renal failure patients showed morphological abnormalities at the time of AVF construction. This may influence the outcome of shunts in terms of future stenosis and failure.     

Contribution of proliferating leukocytes to phenotypic change in smooth muscle cells during the development of coronary arteriosclerosis in transplanted hearts

BACKGROUND: It is well known that coronary arteriosclerosis after heart transplantation is concentric and rich in smooth muscle cells (SMCs); however, the role played by rejection in the intimal thickening caused by SMCs in coronary arteriosclerosis remains unclear. In this study, we examined the process of intimal hyperplasia caused by SMCs and evaluated the relationship between the differentiation state of SMCs and local inflammation caused by rejection. METHODS: Lewis rat hearts were heterotopically transplanted into F344 rats (allotransplantation group) or other Lewis rats (isotransplantation group). Cyclosporin A (5 mg/kg/day) was injected intramuscularly for 20 days after transplantation in both groups. The transplanted hearts were examined immunohistochemically using several monoclonal antibodies; namely, HHF-35, CGA7, vimentin, alpha-actin, HIS36, R73 and proliferating cell nuclear antigen (PCNA). To evaluate the degree of local immunological response caused by rejection, the anti-PCNA antibody was used. To reveal the subtypes of proliferating cells in the thickened intima, HIS36 and R73 antibodies were used. RESULTS: In the allotransplantation group, SMCs in the media began to undergo a phenotypic change toward a poorly differentiated state 30 days after transplantation. Intimal hyperplasia was observed 60 days after transplantation, the thickened intima being composed mainly of dedifferentiated SMCs with abundant PCNA(+), most of which were macrophages and T cells. The state of differentiation of SMCs in the thickened intima 90 days after transplantation varied from a dedifferentiated to a highly differentiated state. These changes were strongly correlated with the expression of PCNA. CONCLUSION: The expression of PCNA was strongly correlated with the differentiation state of SMCs. Thus, local inflammation caused by rejection may play an important role in the initiation of phenotypic change in SMCs.     

透明质酸栓塞对动脉壁结构以及动脉血中IL-1、VEGF和MMP9表达的影响

探讨透明质酸栓塞后对动脉壁的影响，以及对白介素1（I L - 1）、基质金属蛋白 酶9（MMP9）和血管内皮生长因子（VEGF）的表达差异。方法 将9只白兔采用随机数字表法分为对照 组、HA组和结扎组，各3例。对照组注射生理盐水，HA组注射透明质酸栓塞血管，结扎组使用外科缝 线结扎动脉血管阻断血管血流。术前、注射后1、3、5 d抽取各组兔耳中央动脉栓塞部位远心端血 液5 ml，实时荧光PCR法检测其动脉血中IL-1、MMP9和VEGF表达水平，并进行组间比较。第5天终止实 验，处死所有白兔，取兔耳中央动脉血管壁经HE染色后在显微镜下观察。结果 QPCR结果显示：结 扎组与HA组IL-1、MMP9和VEGF的表达水平均高于对照组（P ＜0.05）；结扎组IL-1、MMP9和VEGF 表达水平均高于HA组（P ＜0.05）。HE染色结果显示：对照组动脉壁结构完整；HA组有轻微内膜增 生；结扎组内膜增生严重，平滑肌层紊乱。结论 透明质酸注射栓塞血管或动脉结扎血管均可引起动 脉血IL-1、MMP9和VEGF表达水平升高，引起动脉壁重塑，但透明质酸注射引起的栓塞较结扎动脉对 IL-1、MMP9和VEGF表达水平及动脉壁重塑的影响更小。