Sun Chun-yan, Hu Yu, Wang Hua-fang, He Wen-juan, Wang Ya-dan, Wu Tao
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Chin Med J (Engl). 2006 Apr 5;119(7):589-95.
Recent studies have proved that brain-derived neurotrophic factor (BDNF) possesses angiogenic activity in vitro and in vivo. However, the proangiogenic mechanism of BDNF has not yet been provided with enough information. To explore the proangiogenic mechanism of BDNF, we investigated the effects of BDNF on extracellular proteolytic enzymes, including matrix metalloproteinases (MMPs) and serine proteases, particularly the urokinase-type plasminogen activator (uPA)-plasmin system in human umbilical vein endothelial cells (HUVECs) model.
Tube formation assay was performed in vitro to evaluate the effects of BDNF on angiogenesis. The HUVECs were treated with various concentrations of BDNF (25 - 400 ng/ml) for different (6 - 48 hours), reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assay MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA in HUVECs, and the conditioned medium was analyzed for MMP and uPA activity by gelatin zymography and fibrin zymography, respectively. uPA, plasminogen activator inhibitor (PAI)-1, tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-2 were quantified by western blotting analysis.
BDNF elicited robust and elongated angiogeneis in two-dimensional cultures of HUVECs in comparison with control. The stimulation of serum-starved HUVECs with BDNF caused obvious increase in MMP-2 and MMP-9 mRNA expression and induced the pro-MMP-2 and pro-MMP-9 activation without significant differences in proliferation. However, BDNF had no effect on TIMP-1 and TIMP-2 production. BDNF increased uPA and PAI-1 production in a dose-dependent manner. Maximal activation of uPA and PAI-1 expression in HUVECs was induced by 100 ng/ml BDNF, while effects of 200 ng/ml and 400 ng/ml BDNF were slightly reduced in comparison with with those of 100 ng/ml. Protease activity for uPA was also increased by BDNF in a dose-dependent manner. BDNF also stimulated uPA and PAI-1 production beyond that in control cultures in a time-dependent manner from 12 hours to 48 hours after BDNF treatment.
BDNF stimulates MMP and uPA/PAI-1 proteolytic network in HUVECs, which may be important to the acquisition of proangiogenic potential.
最近的研究证明，脑源性神经营养因子（BDNF）在体外和体内均具有血管生成活性。然而，BDNF的促血管生成机制尚未得到充分的研究。为了探究BDNF的促血管生成机制，我们在人脐静脉内皮细胞（HUVECs）模型中研究了BDNF对细胞外蛋白水解酶的影响，包括基质金属蛋白酶（MMPs）和丝氨酸蛋白酶，特别是尿激酶型纤溶酶原激活剂（uPA）-纤溶酶系统。
进行体外管形成试验以评估BDNF对血管生成的影响。用不同浓度（25 - 400 ng/ml）的BDNF处理HUVECs不同时间（6 - 48小时），采用逆转录聚合酶链反应（RT-PCR）检测HUVECs中MMP-2、MMP-9、TIMP-1和TIMP-2 mRNA的表达，并分别通过明胶酶谱法和纤维蛋白酶谱法分析条件培养基中MMP和uPA的活性。通过蛋白质印迹分析对uPA、纤溶酶原激活剂抑制剂（PAI）-1、金属蛋白酶组织抑制剂（TIMP）-1和TIMP-2进行定量。
与对照组相比，BDNF在HUVECs的二维培养中引发了强大且延长的血管生成。用BDNF刺激血清饥饿的HUVECs导致MMP-2和MMP-9 mRNA表达明显增加，并诱导前MMP-2和前MMP-9的激活，而细胞增殖无显著差异。然而，BDNF对TIMP-1和TIMP-2的产生没有影响。BDNF以剂量依赖性方式增加uPA和PAI-1的产生。100 ng/ml BDNF诱导HUVECs中uPA和PAI-1表达的最大激活，而200 ng/ml和400 ng/ml BDNF的作用与100 ng/ml相比略有降低。BDNF还以剂量依赖性方式增加uPA的蛋白酶活性。BDNF处理后12小时至48小时，BDNF还以时间依赖性方式刺激uPA和PAI-1的产生，使其超过对照培养物中的水平。
BDNF刺激HUVECs中的MMP和uPA/PAI-1蛋白水解网络，这可能对获得促血管生成潜力很重要。
