VEGF和PDGFB受体酪氨酸激酶抑制剂对小鼠角膜新生血管抑制作用的实验研究
|
摘要
正常角膜是透明无血管的,周围血管止于角膜缘。在感染、外伤或免疫反应等病理状态下,毛细血管由角膜缘向角膜内生长,形成角膜新生血管(cornealneovascularization CNV)。虽然新生血管对清除炎症、伤口愈合、抑制角膜融解等有一定促进作用,但角膜新生血管可严重影响视力,破坏角膜正常微环境而使前房相关免疫偏离(anterior chamber-associated immune deviation)消失,从而显著增加角膜移植术后的排斥发生率,排斥率可高达90%。在我国,仅眼外伤所致的CNV就约占整个角膜疾病的10%。就上海地区而言,复旦大学眼耳鼻喉科医院每年所进行的500多例各类角膜手术中,超过2/3的患者有角膜新生血管。另外,还有不少患者因严重的角膜血管化而失去手术机会。虽然科研人员进行了大量的实验与临床研究,角膜新生血管引起的失明及其伴随的角膜移植术后的高排斥率仍然是眼表疾病领域急待解决的棘手问题。
新生血管主要由三部分构成:内皮细胞、周细胞和基底膜。其形成的实质是内皮细胞增殖、迁移和重组的过程。血管发生的步骤为:a)血管内皮细胞、周细胞的活化;b)基底膜的降解;c)活化血管内皮细胞、周细胞的迁移与增生;d)新生毛细血管腔的形成;e)新生毛细血管周边周细胞的出现;f)新生基底膜的形成;g)毛细血管环的形成。
既往的研究表明,在错综复杂的调控机制中,血管内皮生长因子(vascularendothelial growth factor VEGF)被认为是新生血管发生、发展中起关键作用的因子。在病理情况下,VEGF异常高表达,导致其靶细胞血管内皮细胞的增殖和活化。活化的内皮细胞分泌基质金属蛋白酶(matrix metalloproteinase,MMP)等降解基底膜和细胞外基质,使自身游离并开始迁移,从而引起新生血管的出芽生长。因此,各种治疗角膜新生血管的靶点也集中在抑制血管内皮细胞的增殖、迁移方面。多种针对VEGF途径的抗新生血管治疗取得了一定的成效。但是在新生血管形成的晚期,阻断VEGF途径并不能有效抑制已形成的新生血管。由于抗VEGF治疗新生血管的局限性,血管周细胞在血管形成中的作用和意义逐渐为人们所重视。
根据Cursiefen对196例新生血管化角膜的病理研究结果显示,在新生血管形成的2周左右,约80%的新生血管有周细胞包裹。基础研究表明,血管内皮细胞分泌血小板衍生因子-B(PDGFB),而周细胞的胞膜上有PDGFR-β受体。通过PDGFB与受体结合,周细胞被募集在血管内皮细胞周围,与此同时周细胞可分泌VEGF,通过旁分泌的方式作用于血管内皮细胞,以维持血管内皮细胞的稳定。两种细胞相互作用实现“共生”。周细胞的出现,是新生血管“成熟”的标志。在这个阶段,血管内皮细胞对外源性VEGF的依赖大大降低。这也是在新生血管形成的晚期,单纯通过抑制VEGF通路疗效不佳的原因之一。另外,血管内皮细胞和周细胞还通过彼此间直接接触进行调节,通过这种调节,周细胞可对血管生成起控制作用。
因此,通过阻断PDGFB的信号转导,抑制血管周细胞在内皮细胞外的集聚,使周细胞与内皮细胞失去接触,失去周细胞支持的内皮细胞,一旦失去VEGF的刺激将发生细胞凋亡,而最终导致新生血管退化。这个推论在抑制肿瘤新生血管方面得到初步证实。
VEGF和PDGFB通过其相应受体发挥生物学效应。这两类都是跨膜受体,其胞内段有酪氨酸激酶结构域,属于受体酪氨酸激酶(receptor tyrosine kinases,RTKs)超家族。近年来针对酪氨酸激酶这个位点研发出很多颇具临床应用前景的小分子化合物,这些药物通过抑制受体的磷酸化,阻断相应的信号传导,达到抑制VEGF或PDGFB的生物学效应的目的。这些药物在抗肿瘤血管方面取得令人鼓舞的效果,但很少被用来治疗角膜新生血管。本实验选取两种酪氨酸酶抑制剂,针对不同时期小鼠角膜新生血管,采用单纯或联合抑制VEGF和PDGFB信号通路的方法,观察其对角膜新生血管的治疗作用。
目的:观察小鼠角膜新生血管中周细胞出现的时间及形态特点。
方法:通过碱烧伤及机械刮除全角膜上皮的方法制作BALB/C小鼠角膜新生血管的动物模型。于造模后3、7、10、14、21和28天,分别处死10只小鼠取全角膜,7片角膜铺片后间接免疫荧光染色,用CD31和Ng2分别标记血管内皮细胞和周细胞,于荧光显微镜或激光共焦显微镜下观察。余角膜组织用于实时RT-PCR检测角膜组织中VEGF,PDGFB和ANGl三种细胞因子mRNA的表达。
结果:造模3天(D3)时,周细胞仅出现于新生血管的基底部,顶部血管裸露;至造模后10天(D10)周细胞包裹至血管顶部,但尖端新生血管芽仍然裸露。造模后14—21天(D14—21),所有血管均见到周细胞。主干血管周细胞呈半球形且排列紧密;分支血管周细胞密度较低,周细胞与周细胞之间有间隙,胞体相对扁平,有伪足样突起,其末梢包绕血管。造模后28天(D28),周细胞密度进一步下降,出现周细胞与血管分离现象。角膜组织中VEGF,PDGFB和ANGlmRNA的表达上调,于造模后10天达峰值。
结论:角膜新生血管形成过程中,周细胞的出现晚于新生血管芽的形成。周细胞密度和形态在主干和分枝血管上存在差异。
第二部分VEGF和PDGFB受体酪氨酸激酶抑制剂对生长期角膜新生血管的抑制作用
目的:比较在角膜新生血管的生长期,单独或联合使用VEGF及PDGFB受体酪氨酸激酶抑制剂对小鼠角膜新生血管的抑制作用。
方法:制作BALB/C小鼠角膜新生血管的动物模型。造模后将小鼠随机分为4组,每组10只。于造模当天腹腔单独注射VEGF、PDGFB受体酪氨酸激酶抑制剂或联合用药,对照为同期小鼠腹腔注射PBS。连续10天后,处死动物取全角膜。7片角膜铺片后间接免疫荧光染色,用CD31和Ng2分别标记血管内皮细胞和周细胞,于荧光显微镜或激光共焦显微镜下观察。余角膜组织用于实时RT-PCR检测VEGF,PDGFB,Angl mRNA的表达。
结果:抗VEGF/VEGFR组,角膜新生血管面积占全角膜面积的百分比由同期对照组的34.35%±9.35%下降到18.95%±4.79%(n=7,p＜0.01)。抗PDGF/PDGFR-β组,角膜新生血管面积占全角膜面积的百分比由同期对照组的34.35%±9.35%下降到22.17%±9.7%(n=7,p＜0.05)。联合用药组,角膜新生血管面积占全角膜面积的百分比由同期对照组的34.35%±9.35%下降到17.16%±5.16%(n=7,p＜0.01)。阻断VEGF/VEGFR或PDGFB/PDGFR-β信号转导通路,对VEGF,PDGFB和ANGlmRNA表达的影响没有统计学差异(P＞0.05)。但联合用药时ANGl的mRNA表达下调(P＜0.05)。
结论:血管生长期阻断VEGF和PDGF的信号传导,均可以有效抑制小鼠角膜新生血管的形成,但以阻断VEGF/VEGFR信号传导效果更显著。联合用药有增效作用。
第三部分VEGF和PDGFB受体酪氨酸激酶抑制剂对稳定期角膜新生血管的抑制作用
目的:比较在血管形成的稳定期,单独或联合使用VEGF及PDGFB受体酪氨酸激酶抑制剂对小鼠角膜新生血管的抑制作用。
方法:制作BALB/C小鼠角膜新生血管的动物模型。于造模后第10天将小鼠随机分为4组,每组10只。腹腔注射VEGF、PDGFB受体酪氨酸激酶抑制剂或联合用药,对照为同期小鼠腹腔注射PBS。连续10天后,处死动物取全角膜。7片角膜铺片后间接免疫荧光染色,用CD31和Ng2分别标记血管内皮细胞和周细胞,于荧光显微镜或激光共焦显微镜下观察。余角膜组织用于实时RT-PCR检测VEGF,PDGFB,Angl mRNA的表达。
结果:抗VEGF/VEGFR组,角膜新生血管面积占全角膜面积的百分比由同期对照组的39.82%±10.13%下降到36.78%±11.93%(n=7,p＞0.05)。抗PDGF/PDGFR-β组,角膜新生血管面积占全角膜面积的百分比由同期对照组的39.82%±10.13%下降到27.23%±10.57%(n=7,p＜0.05)。联合用药组:角膜新生血管面积占全角膜面积的百分比由同期对照组的39.82%±10.13%下降到18.97%±6.62%(n=7,p＜0.01)。阻断PDGF/PDGFR-β信号转导或联合用药,均可显著下调VEGF,PDGFB和ANGlmRNA的表达。
结论:阻断VEGF/VEGFR通路,不能有效抑制稳定期新生血管,而阻断PDGF/PDGF-Rβ信号传导,可以造成稳定期新生血管的退缩;联合用药有增效作用。
Physiologically avascular cornea is transparent with conjunctiva vessels ending at the limbus.But in numerous pathological processes,such as infection,trauma, immunologic diseases,corneal neovascularization,the formation of new blood vessels from limbus can occur.Although corneal neovasculariztion(CNV) has some advances in reducing inflammation,fasting wound healing,preventing corneal melt,it may significantly alter vision acuity,and worsen the prognosis of subsequent penetrating keratoplasty,which is caused by the loss of the immunologic privilege of the cornea. The rejection risk can reach to 90%.In China,only the CNV caused by trauma occupied 10%of all corneal diseases.In Shanghai area,more than 2/3 of total 500 patients received PKP in Eye & ENT hospital of Fudan University annually suffered from CNV.And some patients even lose the chance to receive the surgery because of severe CNV.The treatment of corneal neovascularizaion remains largely unsuccessful.
Endothelial cells,pericytes and basement are the main components of new vessels. Angiogenesis is a complex processes,including endothelial cells proliferation, migration and remodelling.The major steps are:a) endothelial cells and pericytes activated;b) original basement membrane degradation;c) proliferation and migration of activated endothelial cells and pericytes;d) new vascular tube formation e) recruitment of pericytes;f) new basement deposition;g) vascular netwok formation. Previous studies have disclosed that although multiple stimuli may be involved in the development of new vessels,vascular endothelial growth factor plays a major role in angiogenesis.In pathologic processes,VEGF mRNA expression is up regulated, which stimulates endothelial cells.Active endothelial cells can excrete matrix metalloproteinase(MMPs) to breakdown basement membrane and extracellular matrix(ECM).
Following proteolytic degradation of the ECM,"leader"endothelial cells start to migrate through the degraded matrix.They are followed by proliferating endothelial cells to form blood sprouts.So VEGF has become a therapeutic target.And VEGF blockade has been recently validated as an effective strategy for inhibition of new blood vessel growth.However,several studies have also shown that anti-VEGF therapy may not be so sufficient to cause vessels regression in advanced stage. Because of the limited ability to impact established vessels,the role of pericytes in new vessels formation has been realized gradually.
According to the clinicopathological study on 196 corneal buttons by Cursiefen,2 weeks after onset of CNV,more than 80%of new vessels were covered by pericytes. Basic researches show that PDGF-B is expressed by the sprouting endothelium and PDGFR-βis expressed by the pericyte.An endothelial PDGF-B signal controls pericyte recruitment to angiogenic vessels.Moreover,pericytes release VEGF,which in turn affect endotelial cells survival.The pericyte recruited to vessels means the stability and matuation of new vessels.At this stage,the endothelial cells become less dependent on VEGE This is the reason why interference with VEGF/VEGFR pathway is not effective.Besides the paracrine manner,direct cell-cell contact also enhances the endothelial cells survival.
Based on previous studies,it is reasonable to inhibit PDGFB signaling pathway to cause disruption of pericytes recruitment.Then endothelial cells without pericytes support undergo apoptosis after VEGF withdrawal.This antiangiogenic therapy has been validated in stumor-associated angiogeness vessels.
VEGF and PDGFB proteins bind to their receptors,VEGFR-2 and PDGFR-βrespectively,to stimulate endothelial cells and pericytes proliferation.These are transmembrane receptors with intracellular tyrosine kinase domain(RTK).Recently, some small molecular RTK inhibitors(TKI) have been developed which demonstrate potential therapeutic efficient.These drugs interrupt VEGF/VEGFR or PDGF/PDGFR-βsignaling through inhibition of receptor phosphorylation.These drugs have been used to treat tumor-associated angiogenesis,but seldom applied in corneal neovascularization.In our study,two RTK inhibitors,targeting VEGF/VEGFR and PDGF/PDGFR-β,were used alone or in combination for corneal new vessels.
PartⅠPericyte Recruitment in Mice Corneal Angiogeness
Purpose:To observe the occurrence and morphologic characteristics of pericytes recruitment to corneal new vessels in mice model.
Method:Corneal neovascularization was induced in BALB/C mice by removal of the corneal and limbal epithelium after application of 2ul NaOH(30 mM/l) to the central cornea.At 3,7,10,14,21 and 28 days after scraping,corneas were harvested and immunostained with CD31 for endothelium and Ng2 for pericyte.VEGF,PDGFB and ANG1 mRNA level of corneas were determined by real-time RT-PCR.
Result:3 days after scraping,pericytes only coated to the base of angiogenic sprout. 10 days after scraping,pericytes moved toward the tip direction.14-21 days after scraping,all vascular loops were covered by pericytes.Pericyte density was higher in main trunk than in branch vessel.The pericytes in main trunk exhibited a rounded morphology and closely associated with each other.Others in branch appeared flat shape with long processes wrapping the endothelium.28 days after scraping,the coverage of pericyte decreased,and some pericytes detached from endothelium. VEGF,PDGFB and ANG1 mRNA expression was up-regulated after scraping.
Conclusion:The coverage of corneal new vessels by pericyte lags behined the blood sprout formation.Pericytes demonstrate morphology and density difference in main trunk and branch vessels.
PartⅡThe Therapeutic Effect of VEGF and PDGFB Receptor Tyrosine Kinases inhibitors in Growing Stage of Mice Corneal Angiogenesis
Purpose:To compare using VEGF and PDGFB receptor tyrosine kinase inhibitors alone and in combination for the treatment of growing stage mice corneal new vessels.
Method:Corneal neovascularization was induced in BALB/C mice by removal of the corneal and limbal epithelium after application of 2ul NaOH(30 mM/L) to the central cornea.Either VEGF receptor inhibitorⅡor PDGFB receptor inhibitor or both were administrated by intraperitoneal injection once a day for 10 days.Phosphate-buffered saline(PBS) was used as control.At 10 days after scraping,corneas were harvested and immunostained with CD31 for endothelium and Ng2 for pericyte.VEGF,PDGFB and ANG1 mRNA level of corneas were determined by real-time RT-PCR.
Result:Mice treated with the VEGF,PDGFB receptor inhibitor or both showed an inhibition of corneal neovascularization of 15.4%,12.18%and 17.19%respectively, compared with untreated animals.VEGF,PDGFB and ANG1 mRNA expression was not impaired in the corneas treated with VEGF or PDGFB receptor inhibitor alone. But ANG1 mRNA expression was decreased in corneas treated with both.
Conclusion:Inhibition of VEGF and PDGFB signaling alone was effective to early stage corneal neovascularization.Combination therapy showed more significant efficiency.
PartⅢThe Therapeutic Effect of VEGF and PDGFB Receptor Tyrosine Kinases inhibitors in Maturated Stage of Mice Corneal Angiogenesis
Purpose:To compare using VEGF and PDGFB receptor tyrosine kinase inhibitors alone and in combination for the treatment of maturated stage mice corneal new vessels.
Method:Corneal neovascularization was induced in BALB/C mice by removal of the corneal and limbal epithelium after application of 2ul NaOH(30 mM/L) to the central cornea.At 10 days after scraping,Either VEGF receptor inhibitorⅡor PDGFB receptor inhibitor or both was administrated by intraperitoneal injection once a day for 10 days.Phosphate-buffered saline(PBS) was used as control.At 21 days after scraping,corneas were harvested and immunostained with CD31 for endothelium and Ng2 for pericyte.VEGF,PDGFB and ANG1 mRNA level of corneas were determined by real-time RT-PCR.
Result:Mice treated with the VEGF,PDGFB inhibitor or both showed an inhibition of corneal neovascularization of 3.04%(P＞0.05),12.59%and 20.85%respectively, compared with untreated animals.VEGF,PDGFB and ANG1 mRNA expression was not impaired in the corneas treated with VEGF alone.But VEGF,PDGFB and ANG1 mRNA expression was decreased in corneas treated with PDGF inhibitor alone or both.
Conclusion:Inhibition of VEGF signaling was not effective to maturated stage corneal neovascularization.Inhibition of PDGFB signaling resulted in blood regression.Combination therapy showed more significant efficiency.
新生血管主要由三部分构成:内皮细胞、周细胞和基底膜。其形成的实质是内皮细胞增殖、迁移和重组的过程。血管发生的步骤为:a)血管内皮细胞、周细胞的活化;b)基底膜的降解;c)活化血管内皮细胞、周细胞的迁移与增生;d)新生毛细血管腔的形成;e)新生毛细血管周边周细胞的出现;f)新生基底膜的形成;g)毛细血管环的形成。
既往的研究表明,在错综复杂的调控机制中,血管内皮生长因子(vascularendothelial growth factor VEGF)被认为是新生血管发生、发展中起关键作用的因子。在病理情况下,VEGF异常高表达,导致其靶细胞血管内皮细胞的增殖和活化。活化的内皮细胞分泌基质金属蛋白酶(matrix metalloproteinase,MMP)等降解基底膜和细胞外基质,使自身游离并开始迁移,从而引起新生血管的出芽生长。因此,各种治疗角膜新生血管的靶点也集中在抑制血管内皮细胞的增殖、迁移方面。多种针对VEGF途径的抗新生血管治疗取得了一定的成效。但是在新生血管形成的晚期,阻断VEGF途径并不能有效抑制已形成的新生血管。由于抗VEGF治疗新生血管的局限性,血管周细胞在血管形成中的作用和意义逐渐为人们所重视。
根据Cursiefen对196例新生血管化角膜的病理研究结果显示,在新生血管形成的2周左右,约80%的新生血管有周细胞包裹。基础研究表明,血管内皮细胞分泌血小板衍生因子-B(PDGFB),而周细胞的胞膜上有PDGFR-β受体。通过PDGFB与受体结合,周细胞被募集在血管内皮细胞周围,与此同时周细胞可分泌VEGF,通过旁分泌的方式作用于血管内皮细胞,以维持血管内皮细胞的稳定。两种细胞相互作用实现“共生”。周细胞的出现,是新生血管“成熟”的标志。在这个阶段,血管内皮细胞对外源性VEGF的依赖大大降低。这也是在新生血管形成的晚期,单纯通过抑制VEGF通路疗效不佳的原因之一。另外,血管内皮细胞和周细胞还通过彼此间直接接触进行调节,通过这种调节,周细胞可对血管生成起控制作用。
因此,通过阻断PDGFB的信号转导,抑制血管周细胞在内皮细胞外的集聚,使周细胞与内皮细胞失去接触,失去周细胞支持的内皮细胞,一旦失去VEGF的刺激将发生细胞凋亡,而最终导致新生血管退化。这个推论在抑制肿瘤新生血管方面得到初步证实。
VEGF和PDGFB通过其相应受体发挥生物学效应。这两类都是跨膜受体,其胞内段有酪氨酸激酶结构域,属于受体酪氨酸激酶(receptor tyrosine kinases,RTKs)超家族。近年来针对酪氨酸激酶这个位点研发出很多颇具临床应用前景的小分子化合物,这些药物通过抑制受体的磷酸化,阻断相应的信号传导,达到抑制VEGF或PDGFB的生物学效应的目的。这些药物在抗肿瘤血管方面取得令人鼓舞的效果,但很少被用来治疗角膜新生血管。本实验选取两种酪氨酸酶抑制剂,针对不同时期小鼠角膜新生血管,采用单纯或联合抑制VEGF和PDGFB信号通路的方法,观察其对角膜新生血管的治疗作用。
目的:观察小鼠角膜新生血管中周细胞出现的时间及形态特点。
方法:通过碱烧伤及机械刮除全角膜上皮的方法制作BALB/C小鼠角膜新生血管的动物模型。于造模后3、7、10、14、21和28天,分别处死10只小鼠取全角膜,7片角膜铺片后间接免疫荧光染色,用CD31和Ng2分别标记血管内皮细胞和周细胞,于荧光显微镜或激光共焦显微镜下观察。余角膜组织用于实时RT-PCR检测角膜组织中VEGF,PDGFB和ANGl三种细胞因子mRNA的表达。
结果:造模3天(D3)时,周细胞仅出现于新生血管的基底部,顶部血管裸露;至造模后10天(D10)周细胞包裹至血管顶部,但尖端新生血管芽仍然裸露。造模后14—21天(D14—21),所有血管均见到周细胞。主干血管周细胞呈半球形且排列紧密;分支血管周细胞密度较低,周细胞与周细胞之间有间隙,胞体相对扁平,有伪足样突起,其末梢包绕血管。造模后28天(D28),周细胞密度进一步下降,出现周细胞与血管分离现象。角膜组织中VEGF,PDGFB和ANGlmRNA的表达上调,于造模后10天达峰值。
结论:角膜新生血管形成过程中,周细胞的出现晚于新生血管芽的形成。周细胞密度和形态在主干和分枝血管上存在差异。
第二部分VEGF和PDGFB受体酪氨酸激酶抑制剂对生长期角膜新生血管的抑制作用
目的:比较在角膜新生血管的生长期,单独或联合使用VEGF及PDGFB受体酪氨酸激酶抑制剂对小鼠角膜新生血管的抑制作用。
方法:制作BALB/C小鼠角膜新生血管的动物模型。造模后将小鼠随机分为4组,每组10只。于造模当天腹腔单独注射VEGF、PDGFB受体酪氨酸激酶抑制剂或联合用药,对照为同期小鼠腹腔注射PBS。连续10天后,处死动物取全角膜。7片角膜铺片后间接免疫荧光染色,用CD31和Ng2分别标记血管内皮细胞和周细胞,于荧光显微镜或激光共焦显微镜下观察。余角膜组织用于实时RT-PCR检测VEGF,PDGFB,Angl mRNA的表达。
结果:抗VEGF/VEGFR组,角膜新生血管面积占全角膜面积的百分比由同期对照组的34.35%±9.35%下降到18.95%±4.79%(n=7,p＜0.01)。抗PDGF/PDGFR-β组,角膜新生血管面积占全角膜面积的百分比由同期对照组的34.35%±9.35%下降到22.17%±9.7%(n=7,p＜0.05)。联合用药组,角膜新生血管面积占全角膜面积的百分比由同期对照组的34.35%±9.35%下降到17.16%±5.16%(n=7,p＜0.01)。阻断VEGF/VEGFR或PDGFB/PDGFR-β信号转导通路,对VEGF,PDGFB和ANGlmRNA表达的影响没有统计学差异(P＞0.05)。但联合用药时ANGl的mRNA表达下调(P＜0.05)。
结论:血管生长期阻断VEGF和PDGF的信号传导,均可以有效抑制小鼠角膜新生血管的形成,但以阻断VEGF/VEGFR信号传导效果更显著。联合用药有增效作用。
第三部分VEGF和PDGFB受体酪氨酸激酶抑制剂对稳定期角膜新生血管的抑制作用
目的:比较在血管形成的稳定期,单独或联合使用VEGF及PDGFB受体酪氨酸激酶抑制剂对小鼠角膜新生血管的抑制作用。
方法:制作BALB/C小鼠角膜新生血管的动物模型。于造模后第10天将小鼠随机分为4组,每组10只。腹腔注射VEGF、PDGFB受体酪氨酸激酶抑制剂或联合用药,对照为同期小鼠腹腔注射PBS。连续10天后,处死动物取全角膜。7片角膜铺片后间接免疫荧光染色,用CD31和Ng2分别标记血管内皮细胞和周细胞,于荧光显微镜或激光共焦显微镜下观察。余角膜组织用于实时RT-PCR检测VEGF,PDGFB,Angl mRNA的表达。
结果:抗VEGF/VEGFR组,角膜新生血管面积占全角膜面积的百分比由同期对照组的39.82%±10.13%下降到36.78%±11.93%(n=7,p＞0.05)。抗PDGF/PDGFR-β组,角膜新生血管面积占全角膜面积的百分比由同期对照组的39.82%±10.13%下降到27.23%±10.57%(n=7,p＜0.05)。联合用药组:角膜新生血管面积占全角膜面积的百分比由同期对照组的39.82%±10.13%下降到18.97%±6.62%(n=7,p＜0.01)。阻断PDGF/PDGFR-β信号转导或联合用药,均可显著下调VEGF,PDGFB和ANGlmRNA的表达。
结论:阻断VEGF/VEGFR通路,不能有效抑制稳定期新生血管,而阻断PDGF/PDGF-Rβ信号传导,可以造成稳定期新生血管的退缩;联合用药有增效作用。
Physiologically avascular cornea is transparent with conjunctiva vessels ending at the limbus.But in numerous pathological processes,such as infection,trauma, immunologic diseases,corneal neovascularization,the formation of new blood vessels from limbus can occur.Although corneal neovasculariztion(CNV) has some advances in reducing inflammation,fasting wound healing,preventing corneal melt,it may significantly alter vision acuity,and worsen the prognosis of subsequent penetrating keratoplasty,which is caused by the loss of the immunologic privilege of the cornea. The rejection risk can reach to 90%.In China,only the CNV caused by trauma occupied 10%of all corneal diseases.In Shanghai area,more than 2/3 of total 500 patients received PKP in Eye & ENT hospital of Fudan University annually suffered from CNV.And some patients even lose the chance to receive the surgery because of severe CNV.The treatment of corneal neovascularizaion remains largely unsuccessful.
Endothelial cells,pericytes and basement are the main components of new vessels. Angiogenesis is a complex processes,including endothelial cells proliferation, migration and remodelling.The major steps are:a) endothelial cells and pericytes activated;b) original basement membrane degradation;c) proliferation and migration of activated endothelial cells and pericytes;d) new vascular tube formation e) recruitment of pericytes;f) new basement deposition;g) vascular netwok formation. Previous studies have disclosed that although multiple stimuli may be involved in the development of new vessels,vascular endothelial growth factor plays a major role in angiogenesis.In pathologic processes,VEGF mRNA expression is up regulated, which stimulates endothelial cells.Active endothelial cells can excrete matrix metalloproteinase(MMPs) to breakdown basement membrane and extracellular matrix(ECM).
Following proteolytic degradation of the ECM,"leader"endothelial cells start to migrate through the degraded matrix.They are followed by proliferating endothelial cells to form blood sprouts.So VEGF has become a therapeutic target.And VEGF blockade has been recently validated as an effective strategy for inhibition of new blood vessel growth.However,several studies have also shown that anti-VEGF therapy may not be so sufficient to cause vessels regression in advanced stage. Because of the limited ability to impact established vessels,the role of pericytes in new vessels formation has been realized gradually.
According to the clinicopathological study on 196 corneal buttons by Cursiefen,2 weeks after onset of CNV,more than 80%of new vessels were covered by pericytes. Basic researches show that PDGF-B is expressed by the sprouting endothelium and PDGFR-βis expressed by the pericyte.An endothelial PDGF-B signal controls pericyte recruitment to angiogenic vessels.Moreover,pericytes release VEGF,which in turn affect endotelial cells survival.The pericyte recruited to vessels means the stability and matuation of new vessels.At this stage,the endothelial cells become less dependent on VEGE This is the reason why interference with VEGF/VEGFR pathway is not effective.Besides the paracrine manner,direct cell-cell contact also enhances the endothelial cells survival.
Based on previous studies,it is reasonable to inhibit PDGFB signaling pathway to cause disruption of pericytes recruitment.Then endothelial cells without pericytes support undergo apoptosis after VEGF withdrawal.This antiangiogenic therapy has been validated in stumor-associated angiogeness vessels.
VEGF and PDGFB proteins bind to their receptors,VEGFR-2 and PDGFR-βrespectively,to stimulate endothelial cells and pericytes proliferation.These are transmembrane receptors with intracellular tyrosine kinase domain(RTK).Recently, some small molecular RTK inhibitors(TKI) have been developed which demonstrate potential therapeutic efficient.These drugs interrupt VEGF/VEGFR or PDGF/PDGFR-βsignaling through inhibition of receptor phosphorylation.These drugs have been used to treat tumor-associated angiogenesis,but seldom applied in corneal neovascularization.In our study,two RTK inhibitors,targeting VEGF/VEGFR and PDGF/PDGFR-β,were used alone or in combination for corneal new vessels.
PartⅠPericyte Recruitment in Mice Corneal Angiogeness
Purpose:To observe the occurrence and morphologic characteristics of pericytes recruitment to corneal new vessels in mice model.
Method:Corneal neovascularization was induced in BALB/C mice by removal of the corneal and limbal epithelium after application of 2ul NaOH(30 mM/l) to the central cornea.At 3,7,10,14,21 and 28 days after scraping,corneas were harvested and immunostained with CD31 for endothelium and Ng2 for pericyte.VEGF,PDGFB and ANG1 mRNA level of corneas were determined by real-time RT-PCR.
Result:3 days after scraping,pericytes only coated to the base of angiogenic sprout. 10 days after scraping,pericytes moved toward the tip direction.14-21 days after scraping,all vascular loops were covered by pericytes.Pericyte density was higher in main trunk than in branch vessel.The pericytes in main trunk exhibited a rounded morphology and closely associated with each other.Others in branch appeared flat shape with long processes wrapping the endothelium.28 days after scraping,the coverage of pericyte decreased,and some pericytes detached from endothelium. VEGF,PDGFB and ANG1 mRNA expression was up-regulated after scraping.
Conclusion:The coverage of corneal new vessels by pericyte lags behined the blood sprout formation.Pericytes demonstrate morphology and density difference in main trunk and branch vessels.
PartⅡThe Therapeutic Effect of VEGF and PDGFB Receptor Tyrosine Kinases inhibitors in Growing Stage of Mice Corneal Angiogenesis
Purpose:To compare using VEGF and PDGFB receptor tyrosine kinase inhibitors alone and in combination for the treatment of growing stage mice corneal new vessels.
Method:Corneal neovascularization was induced in BALB/C mice by removal of the corneal and limbal epithelium after application of 2ul NaOH(30 mM/L) to the central cornea.Either VEGF receptor inhibitorⅡor PDGFB receptor inhibitor or both were administrated by intraperitoneal injection once a day for 10 days.Phosphate-buffered saline(PBS) was used as control.At 10 days after scraping,corneas were harvested and immunostained with CD31 for endothelium and Ng2 for pericyte.VEGF,PDGFB and ANG1 mRNA level of corneas were determined by real-time RT-PCR.
Result:Mice treated with the VEGF,PDGFB receptor inhibitor or both showed an inhibition of corneal neovascularization of 15.4%,12.18%and 17.19%respectively, compared with untreated animals.VEGF,PDGFB and ANG1 mRNA expression was not impaired in the corneas treated with VEGF or PDGFB receptor inhibitor alone. But ANG1 mRNA expression was decreased in corneas treated with both.
Conclusion:Inhibition of VEGF and PDGFB signaling alone was effective to early stage corneal neovascularization.Combination therapy showed more significant efficiency.
PartⅢThe Therapeutic Effect of VEGF and PDGFB Receptor Tyrosine Kinases inhibitors in Maturated Stage of Mice Corneal Angiogenesis
Purpose:To compare using VEGF and PDGFB receptor tyrosine kinase inhibitors alone and in combination for the treatment of maturated stage mice corneal new vessels.
Method:Corneal neovascularization was induced in BALB/C mice by removal of the corneal and limbal epithelium after application of 2ul NaOH(30 mM/L) to the central cornea.At 10 days after scraping,Either VEGF receptor inhibitorⅡor PDGFB receptor inhibitor or both was administrated by intraperitoneal injection once a day for 10 days.Phosphate-buffered saline(PBS) was used as control.At 21 days after scraping,corneas were harvested and immunostained with CD31 for endothelium and Ng2 for pericyte.VEGF,PDGFB and ANG1 mRNA level of corneas were determined by real-time RT-PCR.
Result:Mice treated with the VEGF,PDGFB inhibitor or both showed an inhibition of corneal neovascularization of 3.04%(P＞0.05),12.59%and 20.85%respectively, compared with untreated animals.VEGF,PDGFB and ANG1 mRNA expression was not impaired in the corneas treated with VEGF alone.But VEGF,PDGFB and ANG1 mRNA expression was decreased in corneas treated with PDGF inhibitor alone or both.
Conclusion:Inhibition of VEGF signaling was not effective to maturated stage corneal neovascularization.Inhibition of PDGFB signaling resulted in blood regression.Combination therapy showed more significant efficiency.
引文
1.陈建苏,徐锦堂.角膜新生血管.眼科,2001,10(3):137-143
2.刘祖国,张志红,等.重组人kringle5蛋白滴眼液抑制兔角膜新生血管的研究.中华眼科杂志.2002.38(7):415-8
3.王婷,史伟云.新生血管的防治靶点及在角膜疾病中的研究现状.Int Rev Ophthalmol,2006,30(3):175-179
4.Ambati BK,Nozaki M,Singh N et al.Corneal avascularity is due to soluble VEGF receptor- 1.Nature.2006 Oct 26:443(7114):993-7
5.Kwak N,Okamoto N,Wood JM,et al.VEGF is major stimulator in model of choroidal neovascularization.Invest OphthalmolVisSci2000,41:3158-3186
6.Pierce EA,Avery RL,Foley ED,et al.Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascuarization.Proc Natl Acad Sci USA.1995,92:905-909
7.Adamis ap,Shima DT,Tolention MJ,et al.Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate.Arch Ophthalmol 1996,114:66-71
8.Singh N,Higgins E,Amin S,et al.Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization.Cornea.2007 Jan;26(1):65-72
9.周炼红,邢怡桥,等.角膜内皮生长因子反义核苷酸抑制大鼠新生血管的实验研究.中华眼科杂志.2006.42(5):426-30
10.Singh N,Amin S,Richter E,et al.Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo.Invest Ophthalmol Vis Sci.2005 May;46(5):1647-52
11.Cursiefen C,Hofmann-Rummelt C,Kuchle M,Schlotzer-Schrehardt U.Pericyte recruitment in human corneal angiogenesis:an ultrastructural study with clinicopathological correlation.Br J Ophthalmol.2003 Jan;87(1):101-6
12.Volker Nehls,Elmar Schuchardt and Detlev Drenckhahn.The Effect of Fibroblasts,Vascular Smooth Muscle Cells,and Pericytes on Sprout Formation of Endothelial Cells in a Fibrin Gel Angiogenesis Sy stem.Microvascular Research,1994,48(3):349-363
13.Gabriele Bergers,Steven Song.The role of pericytes in blood-vessel formation and maintenance.Neuro-Oncology,2005(7):452-464
14.Hasumi Y,Ktosowska-Wardega A,Furuhashi M,et al.Identification of a subset of pericytes that respond to combination therapy targeting PDGF and VEGF signaling.Int J Cancer.2007 Dec 15;121(12):2606-14
15.Erber R,Thurnher A,Katsen AD.et al.Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms.FASEB J.2004 Feb;18(2):338-40
16.Gabriele Bergers,Steven Song,Nicole Meyer-Morse,et al.Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors.The Journal of Clinical Investigation,2003,111(9):1287-1295
17.DORIANOF,DAVIDP,A LEXM.Protein tyrosine kinase inhibitors:new treatment modalities.Current Opinion in Pharmacology,20 02,2(4):374-381
18.钟毅,李志裕,尤启冬.血管内皮细胞生长因子受体酪氨酸激酶抑制剂的研究进展.中国新药杂志,2006,15(3):182-184
19.欧杨,傅得兴,郭秀丽.新型多靶点酪氨酸激酶抑制剂苏尼替尼的研究进展.中国新药杂志,2007,16(1):91-94
20.张秀华,林莉萍,丁健.靶向血小板源生长因子受体酪氨酸激酶抑制剂的临床研究进展.中国新药与临床杂志,2006,25(6):454-458
21.Wheatley-Price P,Shepherd FA.Epidermal growth factor receptor inhibitors in the treatment of lung cancer:reality and hopes.Curr Opin Oncol.2008,20(2):162-75
22.Bradley DA,Dunn R,Nanus D,et al.Randomized,double-blind,placebo-controlled phase Ⅱ trial of maintenance sunitinib versus placebo after chemotherapy for patients with advanced urothelial carcinoma:scientific rationale and study design.Clin Genitourin Cancer.2007,5(7):460-3
23.彭珧,张怡轩,郑更新.新型蛋白酪氨酸激酶抑制剂类抗肿瘤药物的研究进展.沈阳药科大学学报.2007,24(7):451-455
24.D'Amato RJ,Loughnan MS,Flynn E,et al.Thalidomide is an inhibitor of angiogenesis.Proc Natl Acad Sci U S A.1994 Apr 26;91(9):4082-5
25.Proia AD,Chandler DB,Haynes WL,et al.Quantitation of corneal neovascularization using computerized image analysis.Lab Invest.1988;58:473-479
26.Balamurali K.Ambati,Akshay Anand,Antonia M.et al Sustained Inhibition of Corneal Neovascularization byGenetic Ablation of CCR5.Investigative Ophthalmology & Visual Science,2003,44(2):590-592
27.Ferrara N,Henzel WJ.Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells.Biochem Biophys Res Commun.1989 Jun 15;161(2):851-8
28.Peter Libby Russell Ross 1929-1999.Nature Medicine.1999,5(5):475
29.Pierce GF,Mustoe TA,Altrock BW et al.Role of platelet-derived growth factor Philos Trans R Soc Lond B Biol Sci.1990 Mar 12;327(1239):155-69..J Cell Biochem.1991 Apr;45(4):319-26
30.Ross R,Bowen-Pope DF,Raines EW.Platelet-derived growth factor and its role in health and disease.Philos Trans R Soc Lond B Biol Sci.1990 Mar 12;327(1239):155-69
31.Schmidt MB,Chen EH,Lynch SE.A review of the effects of insulin-like growth factor and platelet derived growth factor on in vivo cartilage healing and repair.Osteoarthritis Cartilage.2006 May;14(5):403-12
32.朱学军,林再雄,朱益华.等大鼠角膜碱烧伤后血小板源生长因子的表达.眼视光杂志 2004,6(3):169-171
33.Abramsson A,Lindblom P,Betsholtz C.Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors.J Clin Invest.2003,112(8):1142-51
34.Lindblom P,Gerhardt H,Liebner S,et al.Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall.Genes Dev.2003 Aug 1;17(15):1835-40
35.Jain RK,Booth MF.What brings pericytes to tumor vessels? J Clin Invest.2003 Oct;112(8):1134-6
36.艾鹏,谭最.促血管生成素在机体血管形成过程中的作用.国外医学(外科学分),2005,32(1):51-54
37.Claus Cursiefen,Lu Chen,Leonardo P.et al.VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovacularization via macrophage recruitment.J Clin.Invest.2004,113:1040-1050
38.Moromizato Y,Stechschulte S,Miyamoto K,et al.CD18 and ICAM-1-dependent corneal neovascularization and inflammation after limmbal injury.Am J Pathol.2000;157:1277-1281
39.纪小龙,申明识,尹彤 血管周细胞及其肿瘤.临床与实验病理学杂志,2000,16(6):517-519
40.Hellstr_m M,Kal_n M,Lindahl P,Abramsson A,Betsholtz C Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse.Development 1999,126:3047-3055
41.Lindahl P,Hellstrom M,Kalen M,et al.Paracrine PDGF-B/PDGF-R beta signaling controls mesangial cell development in kidney glomeruli.Development.1998,125:3313-3322
42.Leveen P,Pekny M,Gebre-Medhin S,et al.Mice deficient for PDGF B show renal,cardiovascular,and hematological abnormalities.Genes Dev.1994,8:1875-1887
43.Soriano P.Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice.Genes Dev.1994,8:1888-1896
44.Reinmuth N,Liu W,Jung YD.et al.Induction of VEGF in perivascular cells defines a potential paracrine mechanism for endothelial cell survival.FASEB J.2001 May;15(7):1239-41
45.Sundberg C.Kowanetz M,Brown LF.et al.Stable expression of angiopoietin-1and other markers by cultured pericytes:phenotypic similarities to a subpopulation of cells in maturing vessels during later stages of angiogenesis in vivo.Lab Invest.2002.82:387-401
46.Nehls V,Drenckhahn D Heterogeneity of microvascular pericytes for smooth muscle type alpha-actin.J Cell Biol.1991,113:147-154
47.Ozerdem U,Grako KA,Dahlin-Huppe K,et al.NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis.Dev Dyn.2001,222:218-227
48.Bondjers C,Kal_n M,Hellstr_m M,Scheidl SJ,Abramsson A,
49.Renner O,Lindahl P,Cho H,et al.Transcription profiling of PDGF-B deficient embryos identifies RGS5 as a novel marker for pericytes and vascular smooth muscle cells.Am J Pathol.2003,162:721-729
50.Laura E,Benjamin,Itzhak Hemo,et al.A plasticity window for blood vessel remodeling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF.Development.1988,125:1591-1598
51.Sims DE.Recent advances in pericyte biology-implications for health and disease.Can J Cardiol.1991,7:431-443
52.Frank RN,Turczyn TJ,Das A.Pericyte coverage of retinal and cerebral capillaries.Invest Ophthalmol Vis Sci.1990;31(6):999-1007
53.Terman BI,Dougher-Vermazen M,Carrion ME,et al.Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.Biochem Biophys Res Commun.1992 Sep 30;187(3):1579-86
54.Uzui H,Lee JD,Shimizu H,et al.The role of protein-tyrosine phosphorylation and gelatinase production in the migration and proliferation of smooth muscle cells.Atherosclerosis.2000 Mar;149(1):51-9
55.Westermark B,Claesson-Welsh L,Heldin CH.Structural and functional aspects of the receptors for platelet-derived growth factor.Prog Growth Factor Res.1989;1(4):253-66
56.钟利,丁健.血管内皮生长因子受体的信号转导通路.生命的化学.2005,25(2):123-126
57.李娟,李力.视网膜新生血管中受体酪氨酸激酶及其抑制剂的开发利用.中国组织工程研究与临床康复.2007,11(2)359-362
58.Hellstr_m M,Gerhardt H,Kal_n M,et al.Lack of pericytes leads to endothelialhyperplasia and abnormal vascular morphogenesis. J Cell Biol. 2001, 153:543-553
59. Gee MS.Procopio wn,Makonnen S, et al. Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy. Am J Pathol. 2003.162:1883-193
60. Darland DC, D' Amore PA. Blood vessel maturation: vascular development comes of age. J Clin Invest. 1999, 103: 157-158
61. Benjamin LE, Golijania D, Itin A, et al. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest 1999,103:159-165
2.刘祖国,张志红,等.重组人kringle5蛋白滴眼液抑制兔角膜新生血管的研究.中华眼科杂志.2002.38(7):415-8
3.王婷,史伟云.新生血管的防治靶点及在角膜疾病中的研究现状.Int Rev Ophthalmol,2006,30(3):175-179
4.Ambati BK,Nozaki M,Singh N et al.Corneal avascularity is due to soluble VEGF receptor- 1.Nature.2006 Oct 26:443(7114):993-7
5.Kwak N,Okamoto N,Wood JM,et al.VEGF is major stimulator in model of choroidal neovascularization.Invest OphthalmolVisSci2000,41:3158-3186
6.Pierce EA,Avery RL,Foley ED,et al.Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascuarization.Proc Natl Acad Sci USA.1995,92:905-909
7.Adamis ap,Shima DT,Tolention MJ,et al.Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate.Arch Ophthalmol 1996,114:66-71
8.Singh N,Higgins E,Amin S,et al.Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization.Cornea.2007 Jan;26(1):65-72
9.周炼红,邢怡桥,等.角膜内皮生长因子反义核苷酸抑制大鼠新生血管的实验研究.中华眼科杂志.2006.42(5):426-30
10.Singh N,Amin S,Richter E,et al.Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo.Invest Ophthalmol Vis Sci.2005 May;46(5):1647-52
11.Cursiefen C,Hofmann-Rummelt C,Kuchle M,Schlotzer-Schrehardt U.Pericyte recruitment in human corneal angiogenesis:an ultrastructural study with clinicopathological correlation.Br J Ophthalmol.2003 Jan;87(1):101-6
12.Volker Nehls,Elmar Schuchardt and Detlev Drenckhahn.The Effect of Fibroblasts,Vascular Smooth Muscle Cells,and Pericytes on Sprout Formation of Endothelial Cells in a Fibrin Gel Angiogenesis Sy stem.Microvascular Research,1994,48(3):349-363
13.Gabriele Bergers,Steven Song.The role of pericytes in blood-vessel formation and maintenance.Neuro-Oncology,2005(7):452-464
14.Hasumi Y,Ktosowska-Wardega A,Furuhashi M,et al.Identification of a subset of pericytes that respond to combination therapy targeting PDGF and VEGF signaling.Int J Cancer.2007 Dec 15;121(12):2606-14
15.Erber R,Thurnher A,Katsen AD.et al.Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms.FASEB J.2004 Feb;18(2):338-40
16.Gabriele Bergers,Steven Song,Nicole Meyer-Morse,et al.Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors.The Journal of Clinical Investigation,2003,111(9):1287-1295
17.DORIANOF,DAVIDP,A LEXM.Protein tyrosine kinase inhibitors:new treatment modalities.Current Opinion in Pharmacology,20 02,2(4):374-381
18.钟毅,李志裕,尤启冬.血管内皮细胞生长因子受体酪氨酸激酶抑制剂的研究进展.中国新药杂志,2006,15(3):182-184
19.欧杨,傅得兴,郭秀丽.新型多靶点酪氨酸激酶抑制剂苏尼替尼的研究进展.中国新药杂志,2007,16(1):91-94
20.张秀华,林莉萍,丁健.靶向血小板源生长因子受体酪氨酸激酶抑制剂的临床研究进展.中国新药与临床杂志,2006,25(6):454-458
21.Wheatley-Price P,Shepherd FA.Epidermal growth factor receptor inhibitors in the treatment of lung cancer:reality and hopes.Curr Opin Oncol.2008,20(2):162-75
22.Bradley DA,Dunn R,Nanus D,et al.Randomized,double-blind,placebo-controlled phase Ⅱ trial of maintenance sunitinib versus placebo after chemotherapy for patients with advanced urothelial carcinoma:scientific rationale and study design.Clin Genitourin Cancer.2007,5(7):460-3
23.彭珧,张怡轩,郑更新.新型蛋白酪氨酸激酶抑制剂类抗肿瘤药物的研究进展.沈阳药科大学学报.2007,24(7):451-455
24.D'Amato RJ,Loughnan MS,Flynn E,et al.Thalidomide is an inhibitor of angiogenesis.Proc Natl Acad Sci U S A.1994 Apr 26;91(9):4082-5
25.Proia AD,Chandler DB,Haynes WL,et al.Quantitation of corneal neovascularization using computerized image analysis.Lab Invest.1988;58:473-479
26.Balamurali K.Ambati,Akshay Anand,Antonia M.et al Sustained Inhibition of Corneal Neovascularization byGenetic Ablation of CCR5.Investigative Ophthalmology & Visual Science,2003,44(2):590-592
27.Ferrara N,Henzel WJ.Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells.Biochem Biophys Res Commun.1989 Jun 15;161(2):851-8
28.Peter Libby Russell Ross 1929-1999.Nature Medicine.1999,5(5):475
29.Pierce GF,Mustoe TA,Altrock BW et al.Role of platelet-derived growth factor Philos Trans R Soc Lond B Biol Sci.1990 Mar 12;327(1239):155-69..J Cell Biochem.1991 Apr;45(4):319-26
30.Ross R,Bowen-Pope DF,Raines EW.Platelet-derived growth factor and its role in health and disease.Philos Trans R Soc Lond B Biol Sci.1990 Mar 12;327(1239):155-69
31.Schmidt MB,Chen EH,Lynch SE.A review of the effects of insulin-like growth factor and platelet derived growth factor on in vivo cartilage healing and repair.Osteoarthritis Cartilage.2006 May;14(5):403-12
32.朱学军,林再雄,朱益华.等大鼠角膜碱烧伤后血小板源生长因子的表达.眼视光杂志 2004,6(3):169-171
33.Abramsson A,Lindblom P,Betsholtz C.Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors.J Clin Invest.2003,112(8):1142-51
34.Lindblom P,Gerhardt H,Liebner S,et al.Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall.Genes Dev.2003 Aug 1;17(15):1835-40
35.Jain RK,Booth MF.What brings pericytes to tumor vessels? J Clin Invest.2003 Oct;112(8):1134-6
36.艾鹏,谭最.促血管生成素在机体血管形成过程中的作用.国外医学(外科学分),2005,32(1):51-54
37.Claus Cursiefen,Lu Chen,Leonardo P.et al.VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovacularization via macrophage recruitment.J Clin.Invest.2004,113:1040-1050
38.Moromizato Y,Stechschulte S,Miyamoto K,et al.CD18 and ICAM-1-dependent corneal neovascularization and inflammation after limmbal injury.Am J Pathol.2000;157:1277-1281
39.纪小龙,申明识,尹彤 血管周细胞及其肿瘤.临床与实验病理学杂志,2000,16(6):517-519
40.Hellstr_m M,Kal_n M,Lindahl P,Abramsson A,Betsholtz C Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse.Development 1999,126:3047-3055
41.Lindahl P,Hellstrom M,Kalen M,et al.Paracrine PDGF-B/PDGF-R beta signaling controls mesangial cell development in kidney glomeruli.Development.1998,125:3313-3322
42.Leveen P,Pekny M,Gebre-Medhin S,et al.Mice deficient for PDGF B show renal,cardiovascular,and hematological abnormalities.Genes Dev.1994,8:1875-1887
43.Soriano P.Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice.Genes Dev.1994,8:1888-1896
44.Reinmuth N,Liu W,Jung YD.et al.Induction of VEGF in perivascular cells defines a potential paracrine mechanism for endothelial cell survival.FASEB J.2001 May;15(7):1239-41
45.Sundberg C.Kowanetz M,Brown LF.et al.Stable expression of angiopoietin-1and other markers by cultured pericytes:phenotypic similarities to a subpopulation of cells in maturing vessels during later stages of angiogenesis in vivo.Lab Invest.2002.82:387-401
46.Nehls V,Drenckhahn D Heterogeneity of microvascular pericytes for smooth muscle type alpha-actin.J Cell Biol.1991,113:147-154
47.Ozerdem U,Grako KA,Dahlin-Huppe K,et al.NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis.Dev Dyn.2001,222:218-227
48.Bondjers C,Kal_n M,Hellstr_m M,Scheidl SJ,Abramsson A,
49.Renner O,Lindahl P,Cho H,et al.Transcription profiling of PDGF-B deficient embryos identifies RGS5 as a novel marker for pericytes and vascular smooth muscle cells.Am J Pathol.2003,162:721-729
50.Laura E,Benjamin,Itzhak Hemo,et al.A plasticity window for blood vessel remodeling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF.Development.1988,125:1591-1598
51.Sims DE.Recent advances in pericyte biology-implications for health and disease.Can J Cardiol.1991,7:431-443
52.Frank RN,Turczyn TJ,Das A.Pericyte coverage of retinal and cerebral capillaries.Invest Ophthalmol Vis Sci.1990;31(6):999-1007
53.Terman BI,Dougher-Vermazen M,Carrion ME,et al.Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.Biochem Biophys Res Commun.1992 Sep 30;187(3):1579-86
54.Uzui H,Lee JD,Shimizu H,et al.The role of protein-tyrosine phosphorylation and gelatinase production in the migration and proliferation of smooth muscle cells.Atherosclerosis.2000 Mar;149(1):51-9
55.Westermark B,Claesson-Welsh L,Heldin CH.Structural and functional aspects of the receptors for platelet-derived growth factor.Prog Growth Factor Res.1989;1(4):253-66
56.钟利,丁健.血管内皮生长因子受体的信号转导通路.生命的化学.2005,25(2):123-126
57.李娟,李力.视网膜新生血管中受体酪氨酸激酶及其抑制剂的开发利用.中国组织工程研究与临床康复.2007,11(2)359-362
58.Hellstr_m M,Gerhardt H,Kal_n M,et al.Lack of pericytes leads to endothelialhyperplasia and abnormal vascular morphogenesis. J Cell Biol. 2001, 153:543-553
59. Gee MS.Procopio wn,Makonnen S, et al. Tumor vessel development and maturation impose limits on the effectiveness of anti-vascular therapy. Am J Pathol. 2003.162:1883-193
60. Darland DC, D' Amore PA. Blood vessel maturation: vascular development comes of age. J Clin Invest. 1999, 103: 157-158
61. Benjamin LE, Golijania D, Itin A, et al. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest 1999,103:159-165