Emanueli Costanza, Salis Maria B, Van Linthout Sophie, Meloni Marco, Desortes Elisa, Silvestre Jean-Sebastien, Clergue Michel, Figueroa Carlos D, Gadau Sergio, Condorelli Gianluigi, Madeddu Paolo
Experimental Medicine and Gene Therapy Section, INBB, Alghero, Italy.
Circulation. 2004 Sep 21;110(12):1638-44. doi: 10.1161/01.CIR.0000142051.36244.83. Epub 2004 Sep 13.
Angiogenesis gene therapy with human tissue kallikrein (hTK) has shown promise for ischemic disease. The present study was undertaken to (1) assess an optimal gene transfer modality, (2) clarify hTK angiogenic pathways, and (3) discount possible side effects.
The hTK gene was transferred to murine adductors by increasing doses of an adenovirus (Ad.hTK). Heterologous protein production was evaluated by ELISA and immunohistochemistry. Structural and functional characteristics of hTK-induced neovascularization were assessed. Muscular endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF)-A mRNA and protein content were evaluated by real-time polymerase chain reaction and Western blotting. The ability of hTK to phosphorylate-activate Akt/protein kinase B (Akt-B) and VEGF receptor 2 (VEGF-R2) was also determined. Implication of the aforementioned mechanisms in Ad.hTK-induced neovascularization was challenged by blocking Akt-B with a dominant-negative Akt construct; NOS with N(G)-nitro-L-arginine methyl ester; and VEGF-A with neutralizing antibody, VEGF-R2 antagonist, or Ad carrying soluble VEGF-R1 gene. We found that 10(7) PFU Ad.hTK led to peak increases in capillary and arteriole density. Newly developed arterioles persisted for up to 8 weeks. Ad.hTK did not change microvascular permeability. Ad.hTK upregulated eNOS mRNA and protein and activated Akt-B through Ser-473 phosphorylation. Inhibitory studies documented that these biochemical events were instrumental to Ad.hTK-induced neovascularization. In contrast, Ad.hTK neither affected VEGF-A and VEGF-R2 levels nor increased VEGF-R2 phosphorylation. Consistently, Ad.hTK-induced neovascularization was not disturbed by any of the different approaches used to block VEGF-A.
Our findings provide new information on the pathway involved in hTK-induced neoangiogenesis and represent an advancement toward clinical applications with Ad.hTK.
用人组织激肽释放酶（hTK）进行血管生成基因治疗已显示出对缺血性疾病的治疗前景。本研究旨在：（1）评估最佳基因转移方式；（2）阐明hTK血管生成途径；（3）排除可能的副作用。
通过增加腺病毒（Ad.hTK）剂量将hTK基因转移至小鼠内收肌。采用酶联免疫吸附测定（ELISA）和免疫组织化学评估异源蛋白产生情况。评估hTK诱导的新生血管形成的结构和功能特征。通过实时聚合酶链反应和蛋白质印迹法评估肌肉内皮型一氧化氮合酶（eNOS）和血管内皮生长因子（VEGF）-A的mRNA和蛋白含量。还测定了hTK磷酸化激活Akt/蛋白激酶B（Akt-B）和VEGF受体2（VEGF-R2）的能力。用显性负性Akt构建体阻断Akt-B；用N(G)-硝基-L-精氨酸甲酯阻断一氧化氮合酶；用中和抗体、VEGF-R2拮抗剂或携带可溶性VEGF-R1基因的腺病毒阻断VEGF-A，以验证上述机制在Ad.hTK诱导的新生血管形成中的作用。我们发现10(7)个病毒粒子单位（PFU）的Ad.hTK可使毛细血管和小动脉密度达到峰值增加。新形成的小动脉可持续长达8周。Ad.hTK未改变微血管通透性。Ad.hTK上调eNOS的mRNA和蛋白水平，并通过丝氨酸473磷酸化激活Akt-B。抑制性研究表明这些生化事件对Ad.hTK诱导的新生血管形成至关重要。相比之下，Ad.hTK既不影响VEGF-A和VEGF-R2水平，也不增加VEGF-R2磷酸化。一致地，Ad.hTK诱导的新生血管形成未受到用于阻断VEGF-A的任何不同方法的干扰。
我们的研究结果提供了关于hTK诱导新生血管形成所涉及途径的新信息，并代表了Ad.hTK临床应用方面的进展。
