肌肉内血管畸形侵袭和凋亡的病理生物学研究
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摘要
第一部分肌肉内血管畸形组织病理研究及MMP-9和磷酸化Tie2的表达
目的明确肌肉内血管畸形(IMVM)病理特点并检测组织中基质金属蛋白酶-9(MMP-9)及磷酸化的Tie2(P-Tie2)的表达,分析临床表现与病理改变之间的相关性,探讨其意义。
方法收集临床肌肉血管畸形标本39例、正常颅面部皮下组织标本10例;采用H.E.染色观察IMVM病理组织形态学改变,ABC免疫组化染色检测MMP-9、P-Tie2的表达。图像分析软件比较染色强度变化。
结果IMVM病理镜下表现为大小不一的畸形血管腔聚集成团或散落在肌间生长,管壁厚薄不均,可见内皮细胞排列紊乱,伴脂肪组织存在,部分管腔内血栓机化再通,新生毛细血管样结构可长入。MMP-9、P-Tie2在肌肉内血管畸形组织中的阳性表达远高于正常皮下组织中的表达,差异具有统计学意义(P＜0.01);其表达的强度在不同病程、部位、瘤体大小及浸润程度的病例中无明显差异(P＞0.05)。
结论IMVM的病理基础来自其畸形管腔的形态学变化及构成细胞的异常表现;MMP-9、P-Tie2可能参与IMVM的发生、发展,可以成为相对特异性的病理学诊断参考。
第二部分肌肉内血管畸形血管内皮细胞培养、鉴定和生物学行为研究
目的探讨并建立人肌肉内血管畸形内皮细胞(IMVM-EC)体外分离培养的方法;研究IMVM-EC形态、表型和生长、运动、凋亡等生物学行为的特点;比较IMVM-EC同正常人脐静脉内皮细胞(HUVEC)的差异。
方法收集IMVM手术切除后病理组织,运用组织贴壁法培养IMVM-EC,刮除杂细胞进行细胞纯化;收集人剖腹产术后脐带组织,运用Ⅰ型胶原酶消化法培养HUVEC;相差显微镜下观察比较细胞生长过程和形态学改变;细胞免疫荧光和化学染色鉴定两种内皮细胞表面抗原FVⅢ-Ag、CD31、VEGFR、磷酸化的VEGFR(P-VEGFR)、PDGFR、磷酸化的PDGFR(P-PDGFR)的表达;MTT法测定IMVM-EC的生长曲线;将细胞接种于鼠尾胶原观察其管腔形成能力;划痕实验和Transwell实验检测内皮细胞的迁移和侵袭能力;乏血清培养诱导细胞的凋亡模型,流式细胞仪检测不同种细胞的凋亡率。
结果IMVM组织块贴壁3-5天后有EC爬出生长,原代培养3周左右可长满25cm~2培养瓶,呈典型的“铺路石”样细胞形态。IMVM-EC传代后3-6天为对数生长期,有自发性管腔形成现象,4代后开始老化。两种EC皆表达阳性CD31和FVⅢ-Ag。部分血管新生相关的抗原——VEGFR、P-VEGFR、PDGFR、P-PDGFR在IMVM-EC中阴性表达;在HUVEC中弱阳性表达。IMVM-EC和HUVEC具有相似的体外管腔形成能力。IMVM-EC较HUVEC具有增强的细胞迁移能力,且具有突破Matrigel胶模拟的细胞外基质而侵袭生长的能力。IMVM-EC较HUVEC在血清剥夺诱导的调亡模型中凋亡率下降(P＜0.05)。
结论IMVM-EC原代细胞培养模型可以通过组织贴壁法于体外顺利建立。其培养的关键是分辨管腔内皮面种植,刮除杂细胞进行细胞纯化,添加生长因子促进内皮细胞优势性生长。对比HUVEC,IMVM-EC细胞学的异常表现包括:表面部分抗原缺失,管腔形成能力、迁移侵袭能力和对抗凋亡能力增强。IMVM-EC表型和生物学行为的异常是IMVM病理性发生发展的基础之一。
第三部分肌肉内血管畸形侵袭和凋亡相关的分子机制研究
(一) IMVM病理组织中MMP-9、P-tie2的基因和蛋白表达
目的从基因转录和蛋白表达层面检测IMVM组织中MMP-9、P-tie2(Tyr992)的表达,验证两者和IMVM病理学发展的相关性。
方法收集临床手术切除的IMVM病理组织,以正常皮下组织为对照组,通过RT-PCR和Western Blot的方法检测各组织中MMP-9、P-tie2的基因和蛋白表达,以上样标本中的β-actin为内参照,获得灰度比值,统计学比较各组间差异。
结果IMVM组织(n=17)中MMP-9和磷酸化的Tie2的mRNA表达较正常对照组(n=7)明显增高,差异具有统计学意义(P＜0.05);不同部位、治疗次数的MMP-9和Tie2 mRNA的均值比较无明显统计学差异。17例IMVM组织中有13例P-tie2和15例MMP-9的蛋白表达阳性,9例对照组中1例MMP9和P-tie2蛋白表达阳性,其阳性率比较有统计学差异(P＜0.05)。
结论MMP-9和P-Tie2参与IMVM组织病理学改变。
(二) IMVM-EC中MMP-9、P-tie2的基因和蛋白表达和相关信号传导旁路PI3K/Akt的研究
目的从基因转录和蛋白表达层面研究人IMVM细胞中MMP-9、P-tie2(Tyr992)和信号传导通道PI3K/Akt的表达和改变;探讨和IMVM侵袭性发展及凋亡相关的病理学机制。
方法在成功分离和培养IMVM-EC的基础上,运用组织贴壁法培养IMVM来源的血管平滑肌细胞(VSMC)。以IMVM-EC为实验组,VSMC和HUVEC为对照组;乏血清培养诱导内皮细胞凋亡,通过RT-PCR检测不同组内皮细胞中MMP-9和P-tie2的基因表达,统计学分析各组差异。使用药物LY294002和SU11248处理内皮细胞,观察细胞生长改变,Western Blot检测各组细胞中MMP-9、P-tie2、Akt、P-Akt表达的变化,统计学分析比较其差异。
结果IMVM-EC中MMP-9和Tie2的mRNA表达较HUVEC增高,凋亡组和正常组之间表达有差异(P＜0.05)。IMVM来源的VSMC不表达MMP-9和P-tie2蛋白。所有内皮细胞都不同程度地表达Akt蛋白;IMVM-EC正常组和凋亡组表达P-Akt(Ser473),HUVEC阴性表达。PI3k抑制剂LY294002可以阻断IMVM-EC中Akt发生磷酸化,但不阻断P-tie2的表达。抗血管生成药物SU11248随作用浓度增大而下调IMVM-EC和HUVEC表面酪氨酸激酶受体-Tie2的磷酸化表达,并诱发内皮细胞脱壁死亡。
结论高磷酸化的Akt(Ser473)表达和IMVM-EC病理性行为有一定关系。磷酸化的Tie2可能通过活化内皮细胞内信号传导旁路PI3k/Akt来增强IMVM-EC的抗凋亡能力。IMVM-EC通过高表达MMP-9来介导自身增强的侵袭能力。MMP-9抑制剂和特异性的酪氨酸激酶抑制剂有可能成为IMVM生物学治疗的新方向。
(三) P-Akt在IMVM病理组织中的免疫组化研究
目的检测IMVM组织中P-Akt的表达;验证P-Akt和IMVM病理发展的相关性。
方法以正常皮下组织作为对照组,收集IMVM临床病理标本,免疫组化ABC法检测P-Akt的表达;镜下观察对比染色强度的变化,统计学比较阳性率。
结果免疫组织化学染色示39例病理标本中15例阳性表达P-Akt,对照组10例全阴性表达,阳性率比较有统计学差异(P＜0.05)。
结论磷酸化的Akt和IMVM组织病理学发展有一定的相关性。
PART ONE Evaluation of pathological morphology and Expression of matrix metalloproteinase-9 and Phospho-Tie2 in intramuscular vascular malformation
Objective To indentify the character of IMVM pathology;to investigate the expression and significance of matrix metalloproteinase-9(MMP-9)and Phospho-Tie2 (P-tie2) in intramuscular vascular malformation(IMVM) and to analyze the relation between clinical manifestation and pathologic changes.
Methods Specimens of 39 IMVMs and 10 normal craniofacial subcutaneous tissues were collected and stained with H.E and ABC Immunohistochemistry in order to observe the pathological change of the tissue morphology and to detect the expression of MMP-9 and P-tie2.Analysis program on image was adopted to compare the intensity of immunostaining in different samples.
Results Observed under microscope,IMVMs were formed by various abnormal vessels which aggregated together in different sizes,developing within muscles. Accompanied by fat tissue,the endothelial cells arranged disorderly.The thickness of vessel walls varied.Both embolism and recirculation could be found in the lumen with growth of new capillaries.The expression levels of MMP-9 and Phospho-Tie2 were significantly higher in IMVMs than in normal subcutaneous tissues(P＜0.01). Their positive staining intensity had no statistically significant difference among the cases of different courses,locations,dimensions and infiltration degrees(P＞0.05).
Conclusion The morphological abnormity of lumen connections and vascular cells formed the pathological foundation of IMVM.MMP-9 and P-tie2 may be involved in the pathogenesis and development of IMVM and assist the clinical and pathological diagnosis of IMVM.
PART TWO Culture,identification and biological behavior study of endothelial cells from human IMVM
Objective To explore the methods of isolation and cultivation of endothelial cells from IMVM(IMVM-EC);To study the characteristics configuration,phenotype, biological behavior on proliferation,migration,invasion and apoptosis of IMVM-EC; and to analyze its difference from human umbilical vein endothelial cell(HUVEC).
Methods After IMVM specimens and umbilical cords were harvested sterilely, primary explant culure was adopted to isolate and purify IMVM-EC while collagenase digestion was used to culture HUVEC.The growth and morphology of two endothelial cells(ECs) were observed and compared under the inverted phase contrast microscope.FVⅢ-Ag,CD31,VEGFR,Phospho-VEGFR(P-VEGFR),PDGF, and Phospho-PDGFR(P-PDGFR) of cells were identified by immunofluorescence and immunochemistry.Growth curves of IMVM-EC were scaled by MTT assay.ECs were cultured on rat tail collagen to investigate the ability of tube formation.Scratch assay and Transwell test were performed to compare the migration and invasion of two ECs.Cell apoptosis were measured by flow cytometer under the condition of normal culture or FBS starvation.
Results After 3-5 days,ECs could be seen from the margin of primary explants of IMVM.During 3 weeks of primary culturing,IMVM-ECs proliferated and filled the 25cm~2 culture bottle,showing cobblestone monolayer.After passage,IMVM-ECs entered the log phase of the proliferation in 3-6 days and partly represented the shape of tube formations,then aged in 4 passages.Both IMVM-ECs and HUVECs displayed the positive staining of FVⅢ-Ag and CD31.VEGFR、P-VEGFR、PDGFR、P-PDGFR were only slightly positive stained in HUVECs,but not in IMVM-ECs. IMVM-ECs and HUVECs had the similar ability of tube formation in vitro. Compared with HUVECs,IMVM-ECs indicated more powerful capability of migration and invasion,and more resistance to apoptosis either in normal culture condition or in starvation culture.
Conclusion The model of endothelial cell culture from IMVM can be established successfully by using primary explant culture.The key of cultivation is adhesion of endothelium side of explant to the surface of culture bottle,purification of ECs by scraping the useless cells,proliferation of ECs by adding growth factors to the medium.In contrast with HUVECs,IMVM-ECs displayed biological character including lack of some cell surface antigens,enhancement of capacity in tube formation,migration,invasion and resistance to apoptosis.The defects and abnormal behavior of IMVM-EC may accelerate the aggravation of IMVM.
PART THREE Molecular mechanism of invasion and apoptosis in Intramuscular vascular malformation
1.Gene and protein expression of MMP-9 and P-tie2 in IMVM
Objective To investigate the expression of MMP-9,P-tie2(Tyr992) in IMVM tissues from both levels of gene transcription and protein translation and to identify their relation with IMVM pathological development.
Methods Specimens of IMVMs and normal craniofacial subcutaneous tissues were collected.RT-PCR and Western Blot were performed to detect the mRNA and protein expression of MMP-9 and P-tie2 in samples.Expressions ofβ-actin in each specimen were defined as inner controls.The intensities of different expressions were analyzed statistically.
Results The mRNA levels of MMP-9 and P-Tie2 were expressed more highly in IMVM specimens(n=17) than in the comparison group(n=7)(P＜0.05).There were no statistically difference of the mRNA expression in different locations and therapy times.13 of 17 IMVMs' samples showed protein expression of P-tie2 and 15 of 17 showed expression of MMP-9,only 1 of 9 samples of nomal tissues did.The positive ratios were different(P＜0.05).
Conclusions MMP-9 and P-tie2 are involved in the pathological development of IMVM.
2.Study of MMP-9,P-tie2 expression and the related signaling pathway of PI3k/Akt in IMVM-EC
Objective To investigate the expression and change of MMP-9,P-tie2,PI3K/Akt signaling pathway in IMVM-ECs from both levels of gene transcription and protein translation and to explore the pathological molecular mechanism of IMVM in invasion and apoptosis.
Methods On the basis of culture methods of IMVM-EC,the vascular smooth muscle cells(VMSC) from IMVM were isolated and purified by primary explant culturing.Cells were divided into 3 groups:IMVM-EC,HUVEC and VSMC.ECs were induced to apoptosis by starvation culture.RT-PCR was used to detect the mRNA expression of MMP-9 and P-tie2 in different ECs.ECs were treated with LY294002 and SU11248.The expression changes of MMP-9,P-tie2,Akt and P-Akt protein were analyzed by Western Blot.
Results The mRNA levels of MMP-9 and P-Tie2 were expressed more highly in IMVM-ECs than in HUVECs.VSMCs from IMVM didn't show the positive protein expression of MMP-9 and P-tie2.The IMVM-ECs under normal and apoptosis condition indicated the expression of P-Akt,while HUVECs didn't.LY294002 was able to prevent the self-phosphorylation of Akt,but didn't interrupt the expression of P-tie2 in IMVM-ECs.SU11248 could suppress the expression of P-tie2 located in the surface of two ECs and induced their death.
Conclusions Elevated expression of P-Akt(Ser473) may be involved in the pathological behavior of IMVM-ECs.P-tie2 may activate the signaling pathway of PI3k/Akt to improve IMVM-ECs' resistance to apoptosis.IMVM-ECs could express high level of MMP-9 to enhance the invasive capability.Specific inhibitors of MMP-9 and tyrosine kinase on IMVM-ECs are potential to become the selective biological therapy of IMVM.
3.Immunohistochemical study of P-Akt in IMVM
Objective To evaluate the expression and significance of Phospho-Akt(P-Akt, Ser473) in IMVM and identify its relation with IMVM's pathological development.
Methods 39 clinical specimens of IMVM and normal craniofacial subcutaneous tissues were collected and stained with ABC Immunohistochernistry.The positive staining was calculated and analyzed.
Results 15 specimens of 39 IMVM tissues were stained with P-Akt,but the normal tissues(n=10) didn't show the staining.The positive ratios were different(P＜0.05)
Conclusions P-Akt may be involved in the pathological development of IMVM.
目的明确肌肉内血管畸形(IMVM)病理特点并检测组织中基质金属蛋白酶-9(MMP-9)及磷酸化的Tie2(P-Tie2)的表达,分析临床表现与病理改变之间的相关性,探讨其意义。
方法收集临床肌肉血管畸形标本39例、正常颅面部皮下组织标本10例;采用H.E.染色观察IMVM病理组织形态学改变,ABC免疫组化染色检测MMP-9、P-Tie2的表达。图像分析软件比较染色强度变化。
结果IMVM病理镜下表现为大小不一的畸形血管腔聚集成团或散落在肌间生长,管壁厚薄不均,可见内皮细胞排列紊乱,伴脂肪组织存在,部分管腔内血栓机化再通,新生毛细血管样结构可长入。MMP-9、P-Tie2在肌肉内血管畸形组织中的阳性表达远高于正常皮下组织中的表达,差异具有统计学意义(P＜0.01);其表达的强度在不同病程、部位、瘤体大小及浸润程度的病例中无明显差异(P＞0.05)。
结论IMVM的病理基础来自其畸形管腔的形态学变化及构成细胞的异常表现;MMP-9、P-Tie2可能参与IMVM的发生、发展,可以成为相对特异性的病理学诊断参考。
第二部分肌肉内血管畸形血管内皮细胞培养、鉴定和生物学行为研究
目的探讨并建立人肌肉内血管畸形内皮细胞(IMVM-EC)体外分离培养的方法;研究IMVM-EC形态、表型和生长、运动、凋亡等生物学行为的特点;比较IMVM-EC同正常人脐静脉内皮细胞(HUVEC)的差异。
方法收集IMVM手术切除后病理组织,运用组织贴壁法培养IMVM-EC,刮除杂细胞进行细胞纯化;收集人剖腹产术后脐带组织,运用Ⅰ型胶原酶消化法培养HUVEC;相差显微镜下观察比较细胞生长过程和形态学改变;细胞免疫荧光和化学染色鉴定两种内皮细胞表面抗原FVⅢ-Ag、CD31、VEGFR、磷酸化的VEGFR(P-VEGFR)、PDGFR、磷酸化的PDGFR(P-PDGFR)的表达;MTT法测定IMVM-EC的生长曲线;将细胞接种于鼠尾胶原观察其管腔形成能力;划痕实验和Transwell实验检测内皮细胞的迁移和侵袭能力;乏血清培养诱导细胞的凋亡模型,流式细胞仪检测不同种细胞的凋亡率。
结果IMVM组织块贴壁3-5天后有EC爬出生长,原代培养3周左右可长满25cm~2培养瓶,呈典型的“铺路石”样细胞形态。IMVM-EC传代后3-6天为对数生长期,有自发性管腔形成现象,4代后开始老化。两种EC皆表达阳性CD31和FVⅢ-Ag。部分血管新生相关的抗原——VEGFR、P-VEGFR、PDGFR、P-PDGFR在IMVM-EC中阴性表达;在HUVEC中弱阳性表达。IMVM-EC和HUVEC具有相似的体外管腔形成能力。IMVM-EC较HUVEC具有增强的细胞迁移能力,且具有突破Matrigel胶模拟的细胞外基质而侵袭生长的能力。IMVM-EC较HUVEC在血清剥夺诱导的调亡模型中凋亡率下降(P＜0.05)。
结论IMVM-EC原代细胞培养模型可以通过组织贴壁法于体外顺利建立。其培养的关键是分辨管腔内皮面种植,刮除杂细胞进行细胞纯化,添加生长因子促进内皮细胞优势性生长。对比HUVEC,IMVM-EC细胞学的异常表现包括:表面部分抗原缺失,管腔形成能力、迁移侵袭能力和对抗凋亡能力增强。IMVM-EC表型和生物学行为的异常是IMVM病理性发生发展的基础之一。
第三部分肌肉内血管畸形侵袭和凋亡相关的分子机制研究
(一) IMVM病理组织中MMP-9、P-tie2的基因和蛋白表达
目的从基因转录和蛋白表达层面检测IMVM组织中MMP-9、P-tie2(Tyr992)的表达,验证两者和IMVM病理学发展的相关性。
方法收集临床手术切除的IMVM病理组织,以正常皮下组织为对照组,通过RT-PCR和Western Blot的方法检测各组织中MMP-9、P-tie2的基因和蛋白表达,以上样标本中的β-actin为内参照,获得灰度比值,统计学比较各组间差异。
结果IMVM组织(n=17)中MMP-9和磷酸化的Tie2的mRNA表达较正常对照组(n=7)明显增高,差异具有统计学意义(P＜0.05);不同部位、治疗次数的MMP-9和Tie2 mRNA的均值比较无明显统计学差异。17例IMVM组织中有13例P-tie2和15例MMP-9的蛋白表达阳性,9例对照组中1例MMP9和P-tie2蛋白表达阳性,其阳性率比较有统计学差异(P＜0.05)。
结论MMP-9和P-Tie2参与IMVM组织病理学改变。
(二) IMVM-EC中MMP-9、P-tie2的基因和蛋白表达和相关信号传导旁路PI3K/Akt的研究
目的从基因转录和蛋白表达层面研究人IMVM细胞中MMP-9、P-tie2(Tyr992)和信号传导通道PI3K/Akt的表达和改变;探讨和IMVM侵袭性发展及凋亡相关的病理学机制。
方法在成功分离和培养IMVM-EC的基础上,运用组织贴壁法培养IMVM来源的血管平滑肌细胞(VSMC)。以IMVM-EC为实验组,VSMC和HUVEC为对照组;乏血清培养诱导内皮细胞凋亡,通过RT-PCR检测不同组内皮细胞中MMP-9和P-tie2的基因表达,统计学分析各组差异。使用药物LY294002和SU11248处理内皮细胞,观察细胞生长改变,Western Blot检测各组细胞中MMP-9、P-tie2、Akt、P-Akt表达的变化,统计学分析比较其差异。
结果IMVM-EC中MMP-9和Tie2的mRNA表达较HUVEC增高,凋亡组和正常组之间表达有差异(P＜0.05)。IMVM来源的VSMC不表达MMP-9和P-tie2蛋白。所有内皮细胞都不同程度地表达Akt蛋白;IMVM-EC正常组和凋亡组表达P-Akt(Ser473),HUVEC阴性表达。PI3k抑制剂LY294002可以阻断IMVM-EC中Akt发生磷酸化,但不阻断P-tie2的表达。抗血管生成药物SU11248随作用浓度增大而下调IMVM-EC和HUVEC表面酪氨酸激酶受体-Tie2的磷酸化表达,并诱发内皮细胞脱壁死亡。
结论高磷酸化的Akt(Ser473)表达和IMVM-EC病理性行为有一定关系。磷酸化的Tie2可能通过活化内皮细胞内信号传导旁路PI3k/Akt来增强IMVM-EC的抗凋亡能力。IMVM-EC通过高表达MMP-9来介导自身增强的侵袭能力。MMP-9抑制剂和特异性的酪氨酸激酶抑制剂有可能成为IMVM生物学治疗的新方向。
(三) P-Akt在IMVM病理组织中的免疫组化研究
目的检测IMVM组织中P-Akt的表达;验证P-Akt和IMVM病理发展的相关性。
方法以正常皮下组织作为对照组,收集IMVM临床病理标本,免疫组化ABC法检测P-Akt的表达;镜下观察对比染色强度的变化,统计学比较阳性率。
结果免疫组织化学染色示39例病理标本中15例阳性表达P-Akt,对照组10例全阴性表达,阳性率比较有统计学差异(P＜0.05)。
结论磷酸化的Akt和IMVM组织病理学发展有一定的相关性。
PART ONE Evaluation of pathological morphology and Expression of matrix metalloproteinase-9 and Phospho-Tie2 in intramuscular vascular malformation
Objective To indentify the character of IMVM pathology;to investigate the expression and significance of matrix metalloproteinase-9(MMP-9)and Phospho-Tie2 (P-tie2) in intramuscular vascular malformation(IMVM) and to analyze the relation between clinical manifestation and pathologic changes.
Methods Specimens of 39 IMVMs and 10 normal craniofacial subcutaneous tissues were collected and stained with H.E and ABC Immunohistochemistry in order to observe the pathological change of the tissue morphology and to detect the expression of MMP-9 and P-tie2.Analysis program on image was adopted to compare the intensity of immunostaining in different samples.
Results Observed under microscope,IMVMs were formed by various abnormal vessels which aggregated together in different sizes,developing within muscles. Accompanied by fat tissue,the endothelial cells arranged disorderly.The thickness of vessel walls varied.Both embolism and recirculation could be found in the lumen with growth of new capillaries.The expression levels of MMP-9 and Phospho-Tie2 were significantly higher in IMVMs than in normal subcutaneous tissues(P＜0.01). Their positive staining intensity had no statistically significant difference among the cases of different courses,locations,dimensions and infiltration degrees(P＞0.05).
Conclusion The morphological abnormity of lumen connections and vascular cells formed the pathological foundation of IMVM.MMP-9 and P-tie2 may be involved in the pathogenesis and development of IMVM and assist the clinical and pathological diagnosis of IMVM.
PART TWO Culture,identification and biological behavior study of endothelial cells from human IMVM
Objective To explore the methods of isolation and cultivation of endothelial cells from IMVM(IMVM-EC);To study the characteristics configuration,phenotype, biological behavior on proliferation,migration,invasion and apoptosis of IMVM-EC; and to analyze its difference from human umbilical vein endothelial cell(HUVEC).
Methods After IMVM specimens and umbilical cords were harvested sterilely, primary explant culure was adopted to isolate and purify IMVM-EC while collagenase digestion was used to culture HUVEC.The growth and morphology of two endothelial cells(ECs) were observed and compared under the inverted phase contrast microscope.FVⅢ-Ag,CD31,VEGFR,Phospho-VEGFR(P-VEGFR),PDGF, and Phospho-PDGFR(P-PDGFR) of cells were identified by immunofluorescence and immunochemistry.Growth curves of IMVM-EC were scaled by MTT assay.ECs were cultured on rat tail collagen to investigate the ability of tube formation.Scratch assay and Transwell test were performed to compare the migration and invasion of two ECs.Cell apoptosis were measured by flow cytometer under the condition of normal culture or FBS starvation.
Results After 3-5 days,ECs could be seen from the margin of primary explants of IMVM.During 3 weeks of primary culturing,IMVM-ECs proliferated and filled the 25cm~2 culture bottle,showing cobblestone monolayer.After passage,IMVM-ECs entered the log phase of the proliferation in 3-6 days and partly represented the shape of tube formations,then aged in 4 passages.Both IMVM-ECs and HUVECs displayed the positive staining of FVⅢ-Ag and CD31.VEGFR、P-VEGFR、PDGFR、P-PDGFR were only slightly positive stained in HUVECs,but not in IMVM-ECs. IMVM-ECs and HUVECs had the similar ability of tube formation in vitro. Compared with HUVECs,IMVM-ECs indicated more powerful capability of migration and invasion,and more resistance to apoptosis either in normal culture condition or in starvation culture.
Conclusion The model of endothelial cell culture from IMVM can be established successfully by using primary explant culture.The key of cultivation is adhesion of endothelium side of explant to the surface of culture bottle,purification of ECs by scraping the useless cells,proliferation of ECs by adding growth factors to the medium.In contrast with HUVECs,IMVM-ECs displayed biological character including lack of some cell surface antigens,enhancement of capacity in tube formation,migration,invasion and resistance to apoptosis.The defects and abnormal behavior of IMVM-EC may accelerate the aggravation of IMVM.
PART THREE Molecular mechanism of invasion and apoptosis in Intramuscular vascular malformation
1.Gene and protein expression of MMP-9 and P-tie2 in IMVM
Objective To investigate the expression of MMP-9,P-tie2(Tyr992) in IMVM tissues from both levels of gene transcription and protein translation and to identify their relation with IMVM pathological development.
Methods Specimens of IMVMs and normal craniofacial subcutaneous tissues were collected.RT-PCR and Western Blot were performed to detect the mRNA and protein expression of MMP-9 and P-tie2 in samples.Expressions ofβ-actin in each specimen were defined as inner controls.The intensities of different expressions were analyzed statistically.
Results The mRNA levels of MMP-9 and P-Tie2 were expressed more highly in IMVM specimens(n=17) than in the comparison group(n=7)(P＜0.05).There were no statistically difference of the mRNA expression in different locations and therapy times.13 of 17 IMVMs' samples showed protein expression of P-tie2 and 15 of 17 showed expression of MMP-9,only 1 of 9 samples of nomal tissues did.The positive ratios were different(P＜0.05).
Conclusions MMP-9 and P-tie2 are involved in the pathological development of IMVM.
2.Study of MMP-9,P-tie2 expression and the related signaling pathway of PI3k/Akt in IMVM-EC
Objective To investigate the expression and change of MMP-9,P-tie2,PI3K/Akt signaling pathway in IMVM-ECs from both levels of gene transcription and protein translation and to explore the pathological molecular mechanism of IMVM in invasion and apoptosis.
Methods On the basis of culture methods of IMVM-EC,the vascular smooth muscle cells(VMSC) from IMVM were isolated and purified by primary explant culturing.Cells were divided into 3 groups:IMVM-EC,HUVEC and VSMC.ECs were induced to apoptosis by starvation culture.RT-PCR was used to detect the mRNA expression of MMP-9 and P-tie2 in different ECs.ECs were treated with LY294002 and SU11248.The expression changes of MMP-9,P-tie2,Akt and P-Akt protein were analyzed by Western Blot.
Results The mRNA levels of MMP-9 and P-Tie2 were expressed more highly in IMVM-ECs than in HUVECs.VSMCs from IMVM didn't show the positive protein expression of MMP-9 and P-tie2.The IMVM-ECs under normal and apoptosis condition indicated the expression of P-Akt,while HUVECs didn't.LY294002 was able to prevent the self-phosphorylation of Akt,but didn't interrupt the expression of P-tie2 in IMVM-ECs.SU11248 could suppress the expression of P-tie2 located in the surface of two ECs and induced their death.
Conclusions Elevated expression of P-Akt(Ser473) may be involved in the pathological behavior of IMVM-ECs.P-tie2 may activate the signaling pathway of PI3k/Akt to improve IMVM-ECs' resistance to apoptosis.IMVM-ECs could express high level of MMP-9 to enhance the invasive capability.Specific inhibitors of MMP-9 and tyrosine kinase on IMVM-ECs are potential to become the selective biological therapy of IMVM.
3.Immunohistochemical study of P-Akt in IMVM
Objective To evaluate the expression and significance of Phospho-Akt(P-Akt, Ser473) in IMVM and identify its relation with IMVM's pathological development.
Methods 39 clinical specimens of IMVM and normal craniofacial subcutaneous tissues were collected and stained with ABC Immunohistochernistry.The positive staining was calculated and analyzed.
Results 15 specimens of 39 IMVM tissues were stained with P-Akt,but the normal tissues(n=10) didn't show the staining.The positive ratios were different(P＜0.05)
Conclusions P-Akt may be involved in the pathological development of IMVM.
引文
1.Jochen AW,Anja-A.DB,FolzRR J,et al.Current concepts in the classification,diagnosis and treatment of hemangiomas and vascular malformations of the head and neck.Eur Arch Otorhinolaryngol,2001,258:141-149
2.Carolyn DO,Antonio GN.Endothelial Lesions of Soft Tissues:A Review of Reactive and Neoplastic Entities With Emphasis on Low-Grade Malignant ("Borderline") Vascular Tumors.Advances in Anatomic Pathology,2003,10(2):69-87
3.Frassica FJ,Thompson RC.Evaluation,Diagnosis,and Classification of Benign Soft-Tissue Tumors.J Bone Joint Surg,1996,78-A(1):126-140
4.Rai P,Setia S,Kalra N,et al.Intramuscular vascular malformation of the masseter muscle presenting with turkey wattle sign.Oral surgery Oral medicine oral pathology oral radiology and endodontics,2006,102(5):618-624
5.Damron TA,Beauchamp CP,et al.Soft-tissue lumps and bumps.J Bone and Joint Surg,2003,85(6A):1142-1155
6.Hein KD,Mulliken JB,Kozakewich HPW,et al.Venous Malformations of Skeletal Muscle.Plast Reconstr Surg,2002,110(7):1625-1635
7.Lee BB.Changing Concept on Vascular Malformation:No Longer Enigma.Ann Vase Dis,2008,1(1):11-19
8.Wisniewski SJ,Newcomer K,Stanson AW.Intramuscular Hemangioma of the Foot:A Diagnostic Dilemma.Med Sei Sports Exert,2005,37(10):1655-1657
9.华锦明 郑祖根 唐天驷 等.骨骼肌肌肉内血管瘤.中华骨科杂志,2002,22(10):618-622
10.Lee BB.New Approaches to the Treatment of Congenital Vascular Malformations (CVMs)-A Single Centre Experience.Eur J Vase Endovasc Surg 2005,30:184-197
11.Peter T,Francis JH,Mark CG,et al.Surgical Treatment of Hemangiomas of Soft Tissue.Clin Orthopaedics and related research,2002,399:205-210
12.Gulden A,Serkan Y,Aykut M,et al.Intramasseteric Hemangioma.Plast Reconstr Surg,2002,109(5):1748-1750
13.Wild AT,Krauspe PRR.Hemangioma of skeletal muscle.Arch Orthop Trauma Surg,2000,120:139-143
14.Hohenleutner U,Landthaler M,Hamm H,et al.Hemangiomas of infancy and childhood.JDDG,2007,4(5):334-338
15.O'Hara CD,Nascimento AG.Endothelial Lesions of Soft Tissues:A Review of Reactive and Neoplastic Entities With Emphasis on Low-Grade Malignant ("Borderline") Vascular Tumors.Advances in Anatomic Pathology,2003,10(2):69-87
16.Vikkula M,Boon LM,Carraway kl,et al.Vascular Dysmorphogenesis Caused by an Activating Mutation in the Receptor Tyrosine Kinase TIE2.Cell,1996,87:1181-1190
17.Allen PW,Enzinger FM.Hemangioma of skeletal muscle An analysis of 89 cases.Cancer,1972,29:8-22
18.Brouillard P,Vikkula M.Vascular malformations:localized defects in vascular morphogenesis.Clin Genet,2003,63:340-351
19.Upton J,Coombs CJ,Mulliken JB,et al.Vascular Malformations of the Upper Limb:A Review of 270 Patients.J Hand Surg,1999,24A:1019-1035
20.Lehoux S,Castier Y,Tedgui A.Molecular mechanisms of the vascular responses to haemodynamic forces.J Intern Med,2006,259:381-392
21.Wallace CS,Champion JC,Truskey GA.Adhesion and Function of Human Endothelial Cells Co-cultured on Smooth Muscle Cells.Annals of Biomedical Engineering,2006,35(3):375-386
22.Thorin E,Shatos MA,Shreeve SM,et al.Human Vascular Endothelium Heterogeneity:A Comparative Study of Cerebral and Peripheral Cultured Vascular Endothelial Cells.Stroke,1997,28(2):375-381
23.Tille JC,Pepper MS.Hereditary Vascular Anomalies:New Insights Into Their Pathogenesis.Arterioscler Thromb Vase Biol,2004,24:1578-1590
24.Wang QK.Update on the Molecular Genetics of Vascular Anomalies.Lymphat Res Biol,2005,3(4):226-233.
25.Eerola I,Boon LM,Mulliken JB,et al.Capillary Malformation-Arteriovenous Malformation,a New Clinical and Genetic Disorder Caused by RASA1Mutations.Am J Hum Genet,2003,73:1240-1249
26.Cohen MM.Vascular update:Morphogenesis,tumors,malformations,and molecular dimensions.Am J Med Genet,2006,140A:2013-2038.
27.Hua W,Yan Zh,Shigeaki T,et al.Transformation of vascular endothelial cells by a point mutation in the Tie2 gene from human intramuscular haemangioma.Oncogene,2004,23:8700-8704
28.Eklund L,Olsen BR.Tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling.Experimental Cell Research,2006,312:630 - 641
29.Warda NL,Dumont DJ.The angiopoietins and Tie2/Tek:adding to the complexity of cardiovascular development.Cell and Developmental Biology,2002,13:19-27
30.Wong AL,Haroon,ZA,Wemer S,et al.Tie2 Expression and Phosphorylation in Angiogenic and Quiescent Adult Tissues.Circ Res,1997,81:567-574
31.Ribatti D.Genetic and epigenetic mechanisms in the early development of the vascular system.J Anat,2006,208:139-152
32.Cohen MM.Vasculogenesis,angiogenesis,hemangiomas,and vascular malformations.American Journal of Medical Genetics,2002,108:265-274
33.Takahashi K,Mulliken JB,Harry PW,et al.Cellular markers that distinguish the phase of hemangioma during infancy and childhood.J Clin Invest,1994,93:2357-2364
34.Jennifer JM,Steven JF,Susan MK,et al.Increased Expression of Urinary Matrix Metalloproteinases Parallels the Extent and Activity of Vascular Anomalies.Pediatrics,2005,116(1):38-45
35.陈光忠,李铁林,姜晓丹,等.TGF-β1及MMP2,9在脑动静脉畸形中的表达及意义.中国神经精神疾病杂志,2006,32(1):88-89
36.Godin D,Ivan E,Johnson C,et al.Remodeling of Carotid Artery Is Associated With Increased Expression of Matrix Metalloproteinases in Mouse Blood Flow Cessation Model.Circulation,2000,102:2861-2866
37.Morris PN,Dunmore BJ,Tadros A,et al.Functional analysis of a mutant form of the receptor tyrosine kinase Tie2 causing venous malformations.J Mol Med,2005,83:58-63
38.Morals PN,Dunmore BJ,Brindle NPJ.Mutant Tie2 causing venous malformation signals through She.Biochemical and Biophysical Research Communications,2006,346:335-338
39.Laura MD,Dennis EH,Pengnian CL.Akt is a major angiogenic mediator downstream of the Ang1/Tie2 signaling pathway.Experimental Cell Res,2004,298:167- 177
40.Liekens S,Verbeken E,Vandeputte M,et al.A Novel Animal Model for Hemangiomas:Inhibition of Hemaagioma Development by the Angiogenesis Inhibitor TNP-470.Cancer Research,1999,59:2376-2383
41.Eifert S,Villavicencio JL,Kao TC,et al.Prevalence of deep venous anomalies in congenital vascular malformations of venous predominance.J Vase Surg,2000,31:462-471
42.Fayad L,Hazirolan T,Bluemke D,et al.Vascular malformations in the extremities:emphasis on MR imaging features that guide treatment options.Skeletal Radiol,2006,35:127-137
43.Mackie AS,Kozakewich HPW,Geva T,et al.Vascular Tumors of the Heart in Infants and Children:Case Series and Review of the Literature.Pediatr Cardiol,2005,26:344-349
44.Coussens LM,Fingleton B,Matrisian LM.Matrix metalloproteinase inhibitors and cancer:Trials and tribulations.Science,2002,295:2387-2391
45.Hidalgo M,Eckhardt SG.Development of Matrix Metalloproteinase Inhibitors in Cancer Therapy.J Natl Cancer Inst,2001,93:178-93
46.Baev NI,Awad IA.Endothelial Cell Culture From Human Cerebral Cavernous Malformations.Stroke,1998,29:2426-2434
47.Fiedler U,Krissl T,S Koidl,et al.Angiopoietin-1 and Angiopoietin-2 Share the Same Binding Domains in the Tie-2 Receptor Involving the First Ig-like Loop and the Epidermal Growth Factor-like Repeats.JBC,2003,278(3):1721-1727
48.徐泉,孟保英,党双锁,等.原位RT-PCR检测血管瘤组织Ang2 mRNA及其受体Tie2 mRNA的表达.第四军医大学学报,2004,25(11):979-981
49.Boye E,Yu Y,Paranya G,et al.Clonality and altered behavior of endothelial cells from hemangiomas.J Clin Invest,2001,107:745-752
50.Yu Y,Varughese J,Brown LF,et al.Increased Tie2 Expression,Enhanced Response to Angiopoietin-1,and Dysregulated Angiopoietin-2 Expression in Hemangioma-Derived Endothelial Cells.Am J Pathol,2001,159:2271-2280
51.Tan ST,Hasan Q,Velickovic M,et al.A Novel In Vitro Human Model of Hemangioma.Mod Pathol,2000,13(1):92-99
52.Tang YM,Liu WY,Yu S,et al.A Novel In Vivo Model of Human Hemangioma:Xenograft of Human Hemangioma Tissue on Nude Mice.Plast Reconstr Surg,2007,120:869-878
53.Ema M,Yokomizo T,Wakamatsu A,et al.Primitive erythropoiesis from mesodermal precursors expressing VE-cadherin,PECAM-1,Tie2,endoglin,and CD34 in the mouse embryo.Blood,2006,108:4018-4024
54.Wang DF,Lehman RE,Donner DB,et al.Expression and endocytosis of VEGF and its receptors in human colonic vascular endothelial cells.Am J Physiol Gastrointest Liver Physiol,2002,282:1088-1096
55.Kraling BM,Bischoff J.A simplified method for growth of human microvascular endothelial cells results in decreased senescence and continued responsiveness to cytokines and growth factors.In Vitro Cellular & Developmental Biology,1998,34(4):308-315
56.贾俊,赵怡芳,张文锋,等.人静脉畸形内皮细胞的分离培养和鉴定.中华口腔医学杂志2002,37(4):284-286
57.Auguste P,Javerzat S,Bikfalvi A.Regulation of vascular development by fibroblast growth factors.Cell Tissue Res,2003,314:157-166
58.Toninil T,Rossil F,Claudio PP.Molecular basis of angiogenesis and cancer.Oncogene,2003,22:6549-6556
59.Kameil M,Saunders WB,Bayless KJ,et al.Endothelial tubes assemble from intracellular vacuoles in vivo.Nature,2006,442:453-456
60.Reisa RM,Reis-Filhoa JS,Filho AL,et al.Differential Prox-1 and CD 31expression in mucousae,cutaneous and soft tissue vascular lesions and tumors.Pathology-Research and Practice,2005,201:771-776
61.Uranishi R,Baev NI,Ng PY,et al.Expression of Endothelial Cell Angiogenesis Receptors in Human Cerebrovascular Malformations.Neurosurgery,2001,48:359-368
62.Roskoski R.Sunitinib:A VEGF and PDGF receptor protein kinase and angiogenesis inhibitor.Biochemical and Biophysical Research Communications,2007,356:323-328
63.Ball SG,Shuttleworth CA,Kielty CM.Vascular endothelial growth factor can signal through platelet-derived growth factor receptors.The Journal of Cell Biology,2007,177(3):489-500
64.Shiojima I,Walsh K.Role of Akt Signaling in Vascular Homeostasis and Angiogenesis.Circ Res,2002,90:1243-1250
65.Donovan D,Brown NJ,Bishop ET,et al.Comparison of three in vitro human 'angiogenesis' assays with capillaries formed in vivo.Angiogenesis,2001,4:113-121
66.Gerecht-Nir S,Ziskind A,Cohen S,et al.Human Embryonic Stem Cells as an In Vitro Model for Human Vascular Development and the Induction of Vascular Differentiation. Lab Invest, 2003, 83: 1811-1820
67. Mccloskey KE. Gilroy ME, Nerem RM. Use of Embryonic Stem Cell-Derived Endothelial Cells as a Cell Source to Generate Vessel Structures in Vitro. Tissue Engineering, 2005,11: 497-504
68. Wang H, Chen ShF, Ratner BD, et al. Capillary Differentiation of Endothelial Cells on Microgrooved Surfaces. J Phys Chem C, 2007,111(40): 14602-14606
69. Yamaguchi H, Condeelis J. Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochimica et Biophysica Acta, 2007,1773: 642-652
70. Duval H, Harris M, Li J, et al. New insights into the function and regulation of endothelial cell apoptosis. Angiogenesis, 2003, 6: 171-183
71. Duval H. Johnson N, Li J, et al. Vascular development is disrupted by endothelial cell-specific expression of the anti-apoptotic protein Bcl-2. Angiogenesis, 2007, 10: 55-68
72. Perry B, Banyard J, McLaughlin ER, et al. AKT1 Overexpression in Endothelial Cells Leads to the Development of Cutaneous Vascular Malformations In Vivo. Archives of Dermatology Chicago, 2007,143(4): 504
73. Jackson CJ, Nguyen M. Human Microvascular Endothelial Cells Differ from Macrovascular Endothelial Cells in Their Expression of Matrix Metalloproteinases. J Biochrm Cell Bid, 1997,29: 1167-1177
74. Zaman K, Driscoll R, Hahn D, et al. Monitoring multiple angiogenesis-related molecules in the blood of cancer patients shows a correlation between VEGF-A and MMP-9 levels before treatment and divergent changes after surgical vs. conservative therapy. Int J Cancer, 2006,118: 755-764
75. Chantraina CF, Henriet P, Jodelec S, et al. Mechanisms of pericyte recruitment in tumour angiogenesis: A new role for metalloproteinases. Euro J Cancer, 2006, 42: 310-318
76. Rivilis I, Milkiewicz M, Boyd P, et al. Differential involvement of MMP-2 and VEGF during muscle stretch- versus shear stress-induced angiogenesis. Am J Physiol Heart Circ Physiol, 2002,283: 1430-1438
77. Fiedler U, Scharpfenecker M, Koidl S, et al. The Tie-2 ligand Angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies. Blood, 2004,103: 4150-4156
78. Hegen A, Koidl S, Weindel K, et al. Expression of Angiopoietin-2 in Endothelial Cells Is Controlled by Positive and Negative Regulatory Promoter Elements. Arterioscler Thromb Vasc Biol,2004,24:1803-1809
79.Scharpfenecker M,Fiedler U,Reiss Y,et al.The Tie-2 ligand Angiopoietin-2destabilizes quiescent endothelium through an internal autocrine loop mechanism.Journal of Cell Science,2005,118(4):771-780
80.Arora A,Scholar EM.Role of Tyrosine Kinase Inhibitors in Cancer Therapy.JPET,2005,315:971-979
81.于宝华,周晓燕.P13K/Akt/mTOR的信号传导通路在恶性肿瘤中的研究进展.中华病理学杂志.2005,34(10):674-676
82.Suzuki A,Lu J,Kusakai G,et al.ARK5 Is a Tumor Invasion-Associated Factor Downstream of Akt Signaling.Molecular and cellular biology,2004,24(8):3526-3535
83.Armulik A,Abramsson A,Betsholtz C.Endothelial/Pericyte Interactions.Circ Res,2005,97:512-523
84.Cho A,Graves J,Reidy MA.Mitogen-Aetivated Protein Kinases Mediate Matrix Metalloproteinase-9 Expression in Vascular Smooth Muscle Cells.Arterioscler Thromb Vase Biol,2000,20:2527-2532
85.Hasebe Y,Egawa K,Shibanuma M,et al.Induction of matrix metalloproteinase gene expression in an endothelial cell line by direct interaction with malignant cells.Cancer Sci,2007,98:58-67
86.Dimmeler S,Zeiher AM.Endothelial Cell Apoptosis in Angiogenesis and Vessel Regression.Circ Res,2000,87:434-439
87.Li S,Huang NF,Hsu S.Mechanotransduetion in Endothelial Cell Migration.J Cell Biochem,2005,96:1110-1126
88.Go'rriz E,Carreim JM,Reyes R,et al.Intramuscular low flow vascular malformations:treatment by means of direct percutaneous embolization.European Journal of Radiology,1998,27:161-165
89.Higuera S,Gordley K,Metry DW,et al.Management of Hemangiomas and Pediatric Vascular Malformations.The journal of craniolfacial surgery,2006,17(4):783-789
90.Castro G,Puglisi F,Azambuja E,et al.Angiogenesis and cancer:A cross-talk between basic science and clinical trials(the "do ut des" paradigm).Critical Reviews in Ontology/Hematology,2006,59:40-50
91.Reusch P,Barleon B,Weindel K,et al.Identification of a soluble form of the angiopoietin receptor TIE-2 released from endothelial cells and present in human blood.Angiogenesis,2001,4:123-131
92.Quartarone E,Alonci A,Allegra A,et al.Differential levels of soluble angiopoietin-2 and Tie-2 in patients with haematological malignancies.Eur J Haematol,2006,77:480-485
1.Fletcher BC.Intramuscular angioma:a clinicopathological analysis of 74 cases.Histopathology,1991,18(1):53-59
2.Corstiaan C.Breugem MB,Ch B,et al.Use of Magnetic Resonance Imaging for the Evaluation of Vascular Malformations of the Lower Extremity.Plast Reconstr Surg,2001,108:870
3.Tang P,Hornicek F J,Gebhardt MC,et al.Surgical treatment of hemangiomas of soft tissue.Clinical orthopaedics and related research,2002,399::205-210
4.Eifert S,Villavicencio JL,Kao TC,et al.Prevalence of deep venous anomalies in congenital vascular malformations of venous predominance.J Vasc Surg,2000,31:462-71
5.Rai P,Setia S,Kalra N,et al.Intramuscular vascular malformation of the masseter muscle presenting with turkey wattle sign.Oral surgery Oral medicine oral pathology oral radiology and endodontics,2006,102(5):618-624
6.林晓曦,王炜.体表肿瘤.见:王炜主编.整形外科学.杭州:浙江科学技术出版社,1999,第1版,454-460
7.Lee BB.Changing Concept on Vascular Malformation:No Longer Enigma.Ann Vase Dis,2008,1(1):11-19
8.Lee BB,Yakes W,Kim DI,et al.Management of arteriovenous malformations:A multidisciplinary approach.J Vase Surg,2004,39:590-600
9.Lee BB.Critical Issues in Management of Congenital Vascular Malformation.Ann Vase Surg,2004,18:380-392
10.Fayad L,Hazirolan T,Bluemke D,et al.Vascular malformations in the extremities:emphasis on MR imaging features that guide treatment options.Skeletal Radiol,2006,35:127-137
11.Hein KD,Mulliken JB,Kozakewich HPW,et al.Venous Malformations of Skeletal Muscle.Plast Reconstr Surg,2002,110(7):1625-1635
12.Tille JC,Pepper MS.Hereditary Vascular Anomalies:New Insights Into Their Pathogenesis.Arterioscler Thromb Vase Biol,2004,24:1578-1590
13.Ribatti D.Genetic and epigenetic mechanisms in the early development of the vascular system.J Anat,2006,208:139-152
14.Wang QK.Update on the Molecular Genetics of Vascular Anomalies.Lymphat Res Biol,2005,3(4):226-233.
15.Eerola I,Boon LM,Mulliken JB,et al.Capillary Malformation-Arteriovenous Malformation,a New Clinical and Genetic Disorder Caused by RASA1 Mutations.Am J Hum Genet,2003,73:1240-1249
16.Vikkula M,Boon LM,Can'away kl,et al.Vascular Dysmorphogenesis Caused by an Activating Mutation in the Receptor Tyrosine Kinase TIE2.Cell,1996,87:1181-1190
17.Hua W,Yan Zh,Shigeaki T,et al.Transformation of vascular endothelial cells by a point mutation in the Tie2 gene from human intramuscular haemangioma.Oncogene,2004,23:8700-8704
18.Muhammed U,Ahmet C,et al.Intramuscular Hemangioma of the Temporalis Muscle.J craniofacial surgery,2005,17(3):745-747
19.Lehoux S,Castier Y,Tedgui A.Molecular mechanisms of the vascular responses to haemodynamic forces.J Intern Med,2006,259:381-392
20.Upton J,Coombs C J,Mulliken JB,et al.Vascular Malformations of the Upper Limb:A Review of 270 Patients.J Hand Surg,1999,24A:1019 - 1035
21.Morris PN,Dunmore B J,Tadros A,et al.Functional analysis of a mutant form of the receptor tyrosine kinase Tie2 causing venous malformations.J Mol Med,2005,83:58-63
22.Perry B,Banyard J,McLaughlin ER,Randy Watnick,et al.AKT1 Overexpression in Endothelial Cells Leads to the Development of Cutaneous Vascular Malformations In Vivo.Archives of Dermatology Chicago,2007,143(4):504
23.Cohen MM.Vasculogenesis,angiogenesis,hemangiomas,and vascular malformations.American Journal of Medical Genetics,2002,108:265-274
24.Takahashi K,Mulliken JB,Harry PW,et al.Cellular markers that distinguish the phase of hemangioma during infancy and childhood.J Clin Invest,1994,93:2357-2364
25.Jennifer JM,Steven JF,Susan MK,et al.Increased Expression of Urinary Matrix Metalloproteinases Parallels the Extent and Activity of Vascular Anomalies.Pediatrics,2005,116(1):38-45
26.陈光忠,李铁林,姜晓丹,等.TGF-β1及MMP2,9在脑动静脉畸形中的表达及意义.中国神经精神疾病杂志,2006,32(1):88-89
27.华锦明,郑祖根,唐天驷,等.骨骼肌肌肉内血管瘤.中华骨科杂志,2002,22(10):618-622
28.Jochen AW,Anja-A.DB,FolzRR J,et al.Current concepts in the classification,diagnosis and treatment of hemangiomas and vascular malformations of the head and neck.Eur Arch Otorhinolaryngol,2001,258:141-149
29.Wild AT,Krauspe PRR.Hemangioma of skeletal muscle.Arch Orthop Trauma Surg,2000,120:139-143
30.O'Hara CD,Nascimento ACt Endothelial Lesions of Soft Tissues:A Review of Reactive and Neoplastic Entities With Emphasis on Low-Grade Malignant ("Borderline") Vascular Tumors.Advances in Anatomic Pathology,2003,10(2):69-87
31.Allen PW,Enzinger FM.Hemangioma of skeletal muscle An analysis of 89 cases.Cancer,1972,29:8-22
32.Frassica FJ,Thompson RC.Evaluation,Diagnosis,and Classification of Benign Soft-Tissue Tumors.The Journal of Bone and Joint Surgery,1996,78A(1):126-140
33.Berenguer B,Burrows PE,Zurakowski D,et al.Sclerotherapy of craniofacial venous malformations:complications and results.Plast Reconstr Surg,1999,104:1Y11
34.Sabrina R,Christopher DMF.Angiosarcoma Arising in Hemangioma/Vascular Malformation.Am J Surg Pathol,2002,26(10):1319-1329
35.Cohen MM.Vascular update:Morphogenesis,tumors,malformations,and molecular dimensions.Am J Med Genet,2006,140A:2013-2038.
36.宋慧锋,柴家科,陈敏亮.与体表血管瘤及血管畸形相关的临床综合征.中华整形外科杂志,2006,22(4):314-316
37.Noel AA,Gloviczki P,Cherry KJ,et al.Surgical treatment of venous malformationsin Klippel-Trénaunay syndrome.J Vase Surg,2000,32:840-7
38.Marx MV.SIR 2005 annual meeting film panel case:Klippel-Trenaunay-Weber syndrome.J Vase Interv Radiol,2005,16:1173-1178
39.Lee BB.Advanced management of congenital vascular malformations(CVM).International Angiology,2002,21:209-213
40.Tang P,Homicek F J,Gebhardt MC,et al.Surgical Treatment of Hemangiomas of Soft Tissue.Clinical orthopaedics and related research,2002,399:205-210
41.Yin GQ,Zhang J,Zheng CJ et al.Experimental and Clinical Research on Treatment of Vascular Malformations With Retained Copper Needles.Ann Plast Surg,2008,60:204-208
42.Rockman CB,Rosen RJ,Jacobowitz GR.Transcatheter Embolization of Extremity Vascular Malformations:The Long-term Success of Multiple Interventions.Ann Vase Surg,2003,17:417-423
43.Go'rriz E,Carreira JM,Reyes R,et al.Intramuscular low flow vascular malformations:treatment by means of direct percutaneous embolization.European Journal of Radiology,1998,27:161 - 165
44.Sadanori A,Kozo A,Katsumi T,et al.Therapeutic Choice for Craniofacial Venous Malformations.The journal of crartiolfaeial surgery,2006,17(4):729-735
2.Carolyn DO,Antonio GN.Endothelial Lesions of Soft Tissues:A Review of Reactive and Neoplastic Entities With Emphasis on Low-Grade Malignant ("Borderline") Vascular Tumors.Advances in Anatomic Pathology,2003,10(2):69-87
3.Frassica FJ,Thompson RC.Evaluation,Diagnosis,and Classification of Benign Soft-Tissue Tumors.J Bone Joint Surg,1996,78-A(1):126-140
4.Rai P,Setia S,Kalra N,et al.Intramuscular vascular malformation of the masseter muscle presenting with turkey wattle sign.Oral surgery Oral medicine oral pathology oral radiology and endodontics,2006,102(5):618-624
5.Damron TA,Beauchamp CP,et al.Soft-tissue lumps and bumps.J Bone and Joint Surg,2003,85(6A):1142-1155
6.Hein KD,Mulliken JB,Kozakewich HPW,et al.Venous Malformations of Skeletal Muscle.Plast Reconstr Surg,2002,110(7):1625-1635
7.Lee BB.Changing Concept on Vascular Malformation:No Longer Enigma.Ann Vase Dis,2008,1(1):11-19
8.Wisniewski SJ,Newcomer K,Stanson AW.Intramuscular Hemangioma of the Foot:A Diagnostic Dilemma.Med Sei Sports Exert,2005,37(10):1655-1657
9.华锦明 郑祖根 唐天驷 等.骨骼肌肌肉内血管瘤.中华骨科杂志,2002,22(10):618-622
10.Lee BB.New Approaches to the Treatment of Congenital Vascular Malformations (CVMs)-A Single Centre Experience.Eur J Vase Endovasc Surg 2005,30:184-197
11.Peter T,Francis JH,Mark CG,et al.Surgical Treatment of Hemangiomas of Soft Tissue.Clin Orthopaedics and related research,2002,399:205-210
12.Gulden A,Serkan Y,Aykut M,et al.Intramasseteric Hemangioma.Plast Reconstr Surg,2002,109(5):1748-1750
13.Wild AT,Krauspe PRR.Hemangioma of skeletal muscle.Arch Orthop Trauma Surg,2000,120:139-143
14.Hohenleutner U,Landthaler M,Hamm H,et al.Hemangiomas of infancy and childhood.JDDG,2007,4(5):334-338
15.O'Hara CD,Nascimento AG.Endothelial Lesions of Soft Tissues:A Review of Reactive and Neoplastic Entities With Emphasis on Low-Grade Malignant ("Borderline") Vascular Tumors.Advances in Anatomic Pathology,2003,10(2):69-87
16.Vikkula M,Boon LM,Carraway kl,et al.Vascular Dysmorphogenesis Caused by an Activating Mutation in the Receptor Tyrosine Kinase TIE2.Cell,1996,87:1181-1190
17.Allen PW,Enzinger FM.Hemangioma of skeletal muscle An analysis of 89 cases.Cancer,1972,29:8-22
18.Brouillard P,Vikkula M.Vascular malformations:localized defects in vascular morphogenesis.Clin Genet,2003,63:340-351
19.Upton J,Coombs CJ,Mulliken JB,et al.Vascular Malformations of the Upper Limb:A Review of 270 Patients.J Hand Surg,1999,24A:1019-1035
20.Lehoux S,Castier Y,Tedgui A.Molecular mechanisms of the vascular responses to haemodynamic forces.J Intern Med,2006,259:381-392
21.Wallace CS,Champion JC,Truskey GA.Adhesion and Function of Human Endothelial Cells Co-cultured on Smooth Muscle Cells.Annals of Biomedical Engineering,2006,35(3):375-386
22.Thorin E,Shatos MA,Shreeve SM,et al.Human Vascular Endothelium Heterogeneity:A Comparative Study of Cerebral and Peripheral Cultured Vascular Endothelial Cells.Stroke,1997,28(2):375-381
23.Tille JC,Pepper MS.Hereditary Vascular Anomalies:New Insights Into Their Pathogenesis.Arterioscler Thromb Vase Biol,2004,24:1578-1590
24.Wang QK.Update on the Molecular Genetics of Vascular Anomalies.Lymphat Res Biol,2005,3(4):226-233.
25.Eerola I,Boon LM,Mulliken JB,et al.Capillary Malformation-Arteriovenous Malformation,a New Clinical and Genetic Disorder Caused by RASA1Mutations.Am J Hum Genet,2003,73:1240-1249
26.Cohen MM.Vascular update:Morphogenesis,tumors,malformations,and molecular dimensions.Am J Med Genet,2006,140A:2013-2038.
27.Hua W,Yan Zh,Shigeaki T,et al.Transformation of vascular endothelial cells by a point mutation in the Tie2 gene from human intramuscular haemangioma.Oncogene,2004,23:8700-8704
28.Eklund L,Olsen BR.Tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling.Experimental Cell Research,2006,312:630 - 641
29.Warda NL,Dumont DJ.The angiopoietins and Tie2/Tek:adding to the complexity of cardiovascular development.Cell and Developmental Biology,2002,13:19-27
30.Wong AL,Haroon,ZA,Wemer S,et al.Tie2 Expression and Phosphorylation in Angiogenic and Quiescent Adult Tissues.Circ Res,1997,81:567-574
31.Ribatti D.Genetic and epigenetic mechanisms in the early development of the vascular system.J Anat,2006,208:139-152
32.Cohen MM.Vasculogenesis,angiogenesis,hemangiomas,and vascular malformations.American Journal of Medical Genetics,2002,108:265-274
33.Takahashi K,Mulliken JB,Harry PW,et al.Cellular markers that distinguish the phase of hemangioma during infancy and childhood.J Clin Invest,1994,93:2357-2364
34.Jennifer JM,Steven JF,Susan MK,et al.Increased Expression of Urinary Matrix Metalloproteinases Parallels the Extent and Activity of Vascular Anomalies.Pediatrics,2005,116(1):38-45
35.陈光忠,李铁林,姜晓丹,等.TGF-β1及MMP2,9在脑动静脉畸形中的表达及意义.中国神经精神疾病杂志,2006,32(1):88-89
36.Godin D,Ivan E,Johnson C,et al.Remodeling of Carotid Artery Is Associated With Increased Expression of Matrix Metalloproteinases in Mouse Blood Flow Cessation Model.Circulation,2000,102:2861-2866
37.Morris PN,Dunmore BJ,Tadros A,et al.Functional analysis of a mutant form of the receptor tyrosine kinase Tie2 causing venous malformations.J Mol Med,2005,83:58-63
38.Morals PN,Dunmore BJ,Brindle NPJ.Mutant Tie2 causing venous malformation signals through She.Biochemical and Biophysical Research Communications,2006,346:335-338
39.Laura MD,Dennis EH,Pengnian CL.Akt is a major angiogenic mediator downstream of the Ang1/Tie2 signaling pathway.Experimental Cell Res,2004,298:167- 177
40.Liekens S,Verbeken E,Vandeputte M,et al.A Novel Animal Model for Hemangiomas:Inhibition of Hemaagioma Development by the Angiogenesis Inhibitor TNP-470.Cancer Research,1999,59:2376-2383
41.Eifert S,Villavicencio JL,Kao TC,et al.Prevalence of deep venous anomalies in congenital vascular malformations of venous predominance.J Vase Surg,2000,31:462-471
42.Fayad L,Hazirolan T,Bluemke D,et al.Vascular malformations in the extremities:emphasis on MR imaging features that guide treatment options.Skeletal Radiol,2006,35:127-137
43.Mackie AS,Kozakewich HPW,Geva T,et al.Vascular Tumors of the Heart in Infants and Children:Case Series and Review of the Literature.Pediatr Cardiol,2005,26:344-349
44.Coussens LM,Fingleton B,Matrisian LM.Matrix metalloproteinase inhibitors and cancer:Trials and tribulations.Science,2002,295:2387-2391
45.Hidalgo M,Eckhardt SG.Development of Matrix Metalloproteinase Inhibitors in Cancer Therapy.J Natl Cancer Inst,2001,93:178-93
46.Baev NI,Awad IA.Endothelial Cell Culture From Human Cerebral Cavernous Malformations.Stroke,1998,29:2426-2434
47.Fiedler U,Krissl T,S Koidl,et al.Angiopoietin-1 and Angiopoietin-2 Share the Same Binding Domains in the Tie-2 Receptor Involving the First Ig-like Loop and the Epidermal Growth Factor-like Repeats.JBC,2003,278(3):1721-1727
48.徐泉,孟保英,党双锁,等.原位RT-PCR检测血管瘤组织Ang2 mRNA及其受体Tie2 mRNA的表达.第四军医大学学报,2004,25(11):979-981
49.Boye E,Yu Y,Paranya G,et al.Clonality and altered behavior of endothelial cells from hemangiomas.J Clin Invest,2001,107:745-752
50.Yu Y,Varughese J,Brown LF,et al.Increased Tie2 Expression,Enhanced Response to Angiopoietin-1,and Dysregulated Angiopoietin-2 Expression in Hemangioma-Derived Endothelial Cells.Am J Pathol,2001,159:2271-2280
51.Tan ST,Hasan Q,Velickovic M,et al.A Novel In Vitro Human Model of Hemangioma.Mod Pathol,2000,13(1):92-99
52.Tang YM,Liu WY,Yu S,et al.A Novel In Vivo Model of Human Hemangioma:Xenograft of Human Hemangioma Tissue on Nude Mice.Plast Reconstr Surg,2007,120:869-878
53.Ema M,Yokomizo T,Wakamatsu A,et al.Primitive erythropoiesis from mesodermal precursors expressing VE-cadherin,PECAM-1,Tie2,endoglin,and CD34 in the mouse embryo.Blood,2006,108:4018-4024
54.Wang DF,Lehman RE,Donner DB,et al.Expression and endocytosis of VEGF and its receptors in human colonic vascular endothelial cells.Am J Physiol Gastrointest Liver Physiol,2002,282:1088-1096
55.Kraling BM,Bischoff J.A simplified method for growth of human microvascular endothelial cells results in decreased senescence and continued responsiveness to cytokines and growth factors.In Vitro Cellular & Developmental Biology,1998,34(4):308-315
56.贾俊,赵怡芳,张文锋,等.人静脉畸形内皮细胞的分离培养和鉴定.中华口腔医学杂志2002,37(4):284-286
57.Auguste P,Javerzat S,Bikfalvi A.Regulation of vascular development by fibroblast growth factors.Cell Tissue Res,2003,314:157-166
58.Toninil T,Rossil F,Claudio PP.Molecular basis of angiogenesis and cancer.Oncogene,2003,22:6549-6556
59.Kameil M,Saunders WB,Bayless KJ,et al.Endothelial tubes assemble from intracellular vacuoles in vivo.Nature,2006,442:453-456
60.Reisa RM,Reis-Filhoa JS,Filho AL,et al.Differential Prox-1 and CD 31expression in mucousae,cutaneous and soft tissue vascular lesions and tumors.Pathology-Research and Practice,2005,201:771-776
61.Uranishi R,Baev NI,Ng PY,et al.Expression of Endothelial Cell Angiogenesis Receptors in Human Cerebrovascular Malformations.Neurosurgery,2001,48:359-368
62.Roskoski R.Sunitinib:A VEGF and PDGF receptor protein kinase and angiogenesis inhibitor.Biochemical and Biophysical Research Communications,2007,356:323-328
63.Ball SG,Shuttleworth CA,Kielty CM.Vascular endothelial growth factor can signal through platelet-derived growth factor receptors.The Journal of Cell Biology,2007,177(3):489-500
64.Shiojima I,Walsh K.Role of Akt Signaling in Vascular Homeostasis and Angiogenesis.Circ Res,2002,90:1243-1250
65.Donovan D,Brown NJ,Bishop ET,et al.Comparison of three in vitro human 'angiogenesis' assays with capillaries formed in vivo.Angiogenesis,2001,4:113-121
66.Gerecht-Nir S,Ziskind A,Cohen S,et al.Human Embryonic Stem Cells as an In Vitro Model for Human Vascular Development and the Induction of Vascular Differentiation. Lab Invest, 2003, 83: 1811-1820
67. Mccloskey KE. Gilroy ME, Nerem RM. Use of Embryonic Stem Cell-Derived Endothelial Cells as a Cell Source to Generate Vessel Structures in Vitro. Tissue Engineering, 2005,11: 497-504
68. Wang H, Chen ShF, Ratner BD, et al. Capillary Differentiation of Endothelial Cells on Microgrooved Surfaces. J Phys Chem C, 2007,111(40): 14602-14606
69. Yamaguchi H, Condeelis J. Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochimica et Biophysica Acta, 2007,1773: 642-652
70. Duval H, Harris M, Li J, et al. New insights into the function and regulation of endothelial cell apoptosis. Angiogenesis, 2003, 6: 171-183
71. Duval H. Johnson N, Li J, et al. Vascular development is disrupted by endothelial cell-specific expression of the anti-apoptotic protein Bcl-2. Angiogenesis, 2007, 10: 55-68
72. Perry B, Banyard J, McLaughlin ER, et al. AKT1 Overexpression in Endothelial Cells Leads to the Development of Cutaneous Vascular Malformations In Vivo. Archives of Dermatology Chicago, 2007,143(4): 504
73. Jackson CJ, Nguyen M. Human Microvascular Endothelial Cells Differ from Macrovascular Endothelial Cells in Their Expression of Matrix Metalloproteinases. J Biochrm Cell Bid, 1997,29: 1167-1177
74. Zaman K, Driscoll R, Hahn D, et al. Monitoring multiple angiogenesis-related molecules in the blood of cancer patients shows a correlation between VEGF-A and MMP-9 levels before treatment and divergent changes after surgical vs. conservative therapy. Int J Cancer, 2006,118: 755-764
75. Chantraina CF, Henriet P, Jodelec S, et al. Mechanisms of pericyte recruitment in tumour angiogenesis: A new role for metalloproteinases. Euro J Cancer, 2006, 42: 310-318
76. Rivilis I, Milkiewicz M, Boyd P, et al. Differential involvement of MMP-2 and VEGF during muscle stretch- versus shear stress-induced angiogenesis. Am J Physiol Heart Circ Physiol, 2002,283: 1430-1438
77. Fiedler U, Scharpfenecker M, Koidl S, et al. The Tie-2 ligand Angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies. Blood, 2004,103: 4150-4156
78. Hegen A, Koidl S, Weindel K, et al. Expression of Angiopoietin-2 in Endothelial Cells Is Controlled by Positive and Negative Regulatory Promoter Elements. Arterioscler Thromb Vasc Biol,2004,24:1803-1809
79.Scharpfenecker M,Fiedler U,Reiss Y,et al.The Tie-2 ligand Angiopoietin-2destabilizes quiescent endothelium through an internal autocrine loop mechanism.Journal of Cell Science,2005,118(4):771-780
80.Arora A,Scholar EM.Role of Tyrosine Kinase Inhibitors in Cancer Therapy.JPET,2005,315:971-979
81.于宝华,周晓燕.P13K/Akt/mTOR的信号传导通路在恶性肿瘤中的研究进展.中华病理学杂志.2005,34(10):674-676
82.Suzuki A,Lu J,Kusakai G,et al.ARK5 Is a Tumor Invasion-Associated Factor Downstream of Akt Signaling.Molecular and cellular biology,2004,24(8):3526-3535
83.Armulik A,Abramsson A,Betsholtz C.Endothelial/Pericyte Interactions.Circ Res,2005,97:512-523
84.Cho A,Graves J,Reidy MA.Mitogen-Aetivated Protein Kinases Mediate Matrix Metalloproteinase-9 Expression in Vascular Smooth Muscle Cells.Arterioscler Thromb Vase Biol,2000,20:2527-2532
85.Hasebe Y,Egawa K,Shibanuma M,et al.Induction of matrix metalloproteinase gene expression in an endothelial cell line by direct interaction with malignant cells.Cancer Sci,2007,98:58-67
86.Dimmeler S,Zeiher AM.Endothelial Cell Apoptosis in Angiogenesis and Vessel Regression.Circ Res,2000,87:434-439
87.Li S,Huang NF,Hsu S.Mechanotransduetion in Endothelial Cell Migration.J Cell Biochem,2005,96:1110-1126
88.Go'rriz E,Carreim JM,Reyes R,et al.Intramuscular low flow vascular malformations:treatment by means of direct percutaneous embolization.European Journal of Radiology,1998,27:161-165
89.Higuera S,Gordley K,Metry DW,et al.Management of Hemangiomas and Pediatric Vascular Malformations.The journal of craniolfacial surgery,2006,17(4):783-789
90.Castro G,Puglisi F,Azambuja E,et al.Angiogenesis and cancer:A cross-talk between basic science and clinical trials(the "do ut des" paradigm).Critical Reviews in Ontology/Hematology,2006,59:40-50
91.Reusch P,Barleon B,Weindel K,et al.Identification of a soluble form of the angiopoietin receptor TIE-2 released from endothelial cells and present in human blood.Angiogenesis,2001,4:123-131
92.Quartarone E,Alonci A,Allegra A,et al.Differential levels of soluble angiopoietin-2 and Tie-2 in patients with haematological malignancies.Eur J Haematol,2006,77:480-485
1.Fletcher BC.Intramuscular angioma:a clinicopathological analysis of 74 cases.Histopathology,1991,18(1):53-59
2.Corstiaan C.Breugem MB,Ch B,et al.Use of Magnetic Resonance Imaging for the Evaluation of Vascular Malformations of the Lower Extremity.Plast Reconstr Surg,2001,108:870
3.Tang P,Hornicek F J,Gebhardt MC,et al.Surgical treatment of hemangiomas of soft tissue.Clinical orthopaedics and related research,2002,399::205-210
4.Eifert S,Villavicencio JL,Kao TC,et al.Prevalence of deep venous anomalies in congenital vascular malformations of venous predominance.J Vasc Surg,2000,31:462-71
5.Rai P,Setia S,Kalra N,et al.Intramuscular vascular malformation of the masseter muscle presenting with turkey wattle sign.Oral surgery Oral medicine oral pathology oral radiology and endodontics,2006,102(5):618-624
6.林晓曦,王炜.体表肿瘤.见:王炜主编.整形外科学.杭州:浙江科学技术出版社,1999,第1版,454-460
7.Lee BB.Changing Concept on Vascular Malformation:No Longer Enigma.Ann Vase Dis,2008,1(1):11-19
8.Lee BB,Yakes W,Kim DI,et al.Management of arteriovenous malformations:A multidisciplinary approach.J Vase Surg,2004,39:590-600
9.Lee BB.Critical Issues in Management of Congenital Vascular Malformation.Ann Vase Surg,2004,18:380-392
10.Fayad L,Hazirolan T,Bluemke D,et al.Vascular malformations in the extremities:emphasis on MR imaging features that guide treatment options.Skeletal Radiol,2006,35:127-137
11.Hein KD,Mulliken JB,Kozakewich HPW,et al.Venous Malformations of Skeletal Muscle.Plast Reconstr Surg,2002,110(7):1625-1635
12.Tille JC,Pepper MS.Hereditary Vascular Anomalies:New Insights Into Their Pathogenesis.Arterioscler Thromb Vase Biol,2004,24:1578-1590
13.Ribatti D.Genetic and epigenetic mechanisms in the early development of the vascular system.J Anat,2006,208:139-152
14.Wang QK.Update on the Molecular Genetics of Vascular Anomalies.Lymphat Res Biol,2005,3(4):226-233.
15.Eerola I,Boon LM,Mulliken JB,et al.Capillary Malformation-Arteriovenous Malformation,a New Clinical and Genetic Disorder Caused by RASA1 Mutations.Am J Hum Genet,2003,73:1240-1249
16.Vikkula M,Boon LM,Can'away kl,et al.Vascular Dysmorphogenesis Caused by an Activating Mutation in the Receptor Tyrosine Kinase TIE2.Cell,1996,87:1181-1190
17.Hua W,Yan Zh,Shigeaki T,et al.Transformation of vascular endothelial cells by a point mutation in the Tie2 gene from human intramuscular haemangioma.Oncogene,2004,23:8700-8704
18.Muhammed U,Ahmet C,et al.Intramuscular Hemangioma of the Temporalis Muscle.J craniofacial surgery,2005,17(3):745-747
19.Lehoux S,Castier Y,Tedgui A.Molecular mechanisms of the vascular responses to haemodynamic forces.J Intern Med,2006,259:381-392
20.Upton J,Coombs C J,Mulliken JB,et al.Vascular Malformations of the Upper Limb:A Review of 270 Patients.J Hand Surg,1999,24A:1019 - 1035
21.Morris PN,Dunmore B J,Tadros A,et al.Functional analysis of a mutant form of the receptor tyrosine kinase Tie2 causing venous malformations.J Mol Med,2005,83:58-63
22.Perry B,Banyard J,McLaughlin ER,Randy Watnick,et al.AKT1 Overexpression in Endothelial Cells Leads to the Development of Cutaneous Vascular Malformations In Vivo.Archives of Dermatology Chicago,2007,143(4):504
23.Cohen MM.Vasculogenesis,angiogenesis,hemangiomas,and vascular malformations.American Journal of Medical Genetics,2002,108:265-274
24.Takahashi K,Mulliken JB,Harry PW,et al.Cellular markers that distinguish the phase of hemangioma during infancy and childhood.J Clin Invest,1994,93:2357-2364
25.Jennifer JM,Steven JF,Susan MK,et al.Increased Expression of Urinary Matrix Metalloproteinases Parallels the Extent and Activity of Vascular Anomalies.Pediatrics,2005,116(1):38-45
26.陈光忠,李铁林,姜晓丹,等.TGF-β1及MMP2,9在脑动静脉畸形中的表达及意义.中国神经精神疾病杂志,2006,32(1):88-89
27.华锦明,郑祖根,唐天驷,等.骨骼肌肌肉内血管瘤.中华骨科杂志,2002,22(10):618-622
28.Jochen AW,Anja-A.DB,FolzRR J,et al.Current concepts in the classification,diagnosis and treatment of hemangiomas and vascular malformations of the head and neck.Eur Arch Otorhinolaryngol,2001,258:141-149
29.Wild AT,Krauspe PRR.Hemangioma of skeletal muscle.Arch Orthop Trauma Surg,2000,120:139-143
30.O'Hara CD,Nascimento ACt Endothelial Lesions of Soft Tissues:A Review of Reactive and Neoplastic Entities With Emphasis on Low-Grade Malignant ("Borderline") Vascular Tumors.Advances in Anatomic Pathology,2003,10(2):69-87
31.Allen PW,Enzinger FM.Hemangioma of skeletal muscle An analysis of 89 cases.Cancer,1972,29:8-22
32.Frassica FJ,Thompson RC.Evaluation,Diagnosis,and Classification of Benign Soft-Tissue Tumors.The Journal of Bone and Joint Surgery,1996,78A(1):126-140
33.Berenguer B,Burrows PE,Zurakowski D,et al.Sclerotherapy of craniofacial venous malformations:complications and results.Plast Reconstr Surg,1999,104:1Y11
34.Sabrina R,Christopher DMF.Angiosarcoma Arising in Hemangioma/Vascular Malformation.Am J Surg Pathol,2002,26(10):1319-1329
35.Cohen MM.Vascular update:Morphogenesis,tumors,malformations,and molecular dimensions.Am J Med Genet,2006,140A:2013-2038.
36.宋慧锋,柴家科,陈敏亮.与体表血管瘤及血管畸形相关的临床综合征.中华整形外科杂志,2006,22(4):314-316
37.Noel AA,Gloviczki P,Cherry KJ,et al.Surgical treatment of venous malformationsin Klippel-Trénaunay syndrome.J Vase Surg,2000,32:840-7
38.Marx MV.SIR 2005 annual meeting film panel case:Klippel-Trenaunay-Weber syndrome.J Vase Interv Radiol,2005,16:1173-1178
39.Lee BB.Advanced management of congenital vascular malformations(CVM).International Angiology,2002,21:209-213
40.Tang P,Homicek F J,Gebhardt MC,et al.Surgical Treatment of Hemangiomas of Soft Tissue.Clinical orthopaedics and related research,2002,399:205-210
41.Yin GQ,Zhang J,Zheng CJ et al.Experimental and Clinical Research on Treatment of Vascular Malformations With Retained Copper Needles.Ann Plast Surg,2008,60:204-208
42.Rockman CB,Rosen RJ,Jacobowitz GR.Transcatheter Embolization of Extremity Vascular Malformations:The Long-term Success of Multiple Interventions.Ann Vase Surg,2003,17:417-423
43.Go'rriz E,Carreira JM,Reyes R,et al.Intramuscular low flow vascular malformations:treatment by means of direct percutaneous embolization.European Journal of Radiology,1998,27:161 - 165
44.Sadanori A,Kozo A,Katsumi T,et al.Therapeutic Choice for Craniofacial Venous Malformations.The journal of crartiolfaeial surgery,2006,17(4):729-735