Wary Kishore K, Thakker Geeta D, Humtsoe Joseph O, Yang Jun
Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University System-Health Science Center, 2121 W, Holcombe Blvd, Houston, TX-77030, USA.
Mol Cancer. 2003 Jul 9;2:25. doi: 10.1186/1476-4598-2-25.
Identification of the genes and pathways associated with the activation of endothelial cells (ECs) could help uncover the role of ECs in wound healing, vascular permeability, blood brain barrier function, angiogenesis, diabetic retinopathy, atherosclerosis, psoriasis, and growth of solid tumors.
Herein, we embedded ECs in 3D type I collagen gel, left unstimulated or stimulated with VEGF165, and subjected to suppression subtractive hybridization followed by differential display (SSHDD). Gene fragments obtained from SSHDD were subjected to DNA sequence analysis. Database search with nucleotide sequence were performed using the BLAST algorithm and expression of candidate genes determined by northern blot analysis.
A total of approximately 32 cDNA fragments, including known regulators of angiogenesis, and a set of genes that were not reported to be associated with activation of ECs and angiogenesis previously were identified. We confirmed the mRNA expression of KDR, alpha2 integrin, Stanniocalcin, including a set of 11 candidate genes. Western immunoblotting results indicated that KDR, alpha2 integrin, MMP-1, MMP-2, and VE-cadherin genes were indeed active genes.
We have identified a set of 11 VEGF-responsive endothelial cell candidate genes. Their expression in endothelial cell is confirmed by northern blot analyses. This preliminary report forms as a foundation for functional studies to be performed to reveal their roles in EC activation and pathophysiological events associated with the vasculature including tumor growth.
鉴定与内皮细胞（ECs）激活相关的基因和信号通路，有助于揭示内皮细胞在伤口愈合、血管通透性、血脑屏障功能、血管生成、糖尿病视网膜病变、动脉粥样硬化、银屑病以及实体瘤生长中的作用。
在此，我们将内皮细胞包埋于三维I型胶原凝胶中，不进行刺激或用VEGF165刺激，然后进行抑制性消减杂交及差异显示（SSHDD）。从SSHDD获得的基因片段进行DNA序列分析。使用BLAST算法对核苷酸序列进行数据库搜索，并通过Northern印迹分析确定候选基因的表达。
共鉴定出约32个cDNA片段，包括已知的血管生成调节因子，以及一组先前未报道与内皮细胞激活和血管生成相关的基因。我们证实了KDR、α2整合素、鲟钙蛋白的mRNA表达，包括一组11个候选基因。Western免疫印迹结果表明，KDR、α2整合素、MMP-1、MMP-2和VE-钙黏蛋白基因确实是活性基因。
我们鉴定出一组11个VEGF反应性内皮细胞候选基因。通过Northern印迹分析证实了它们在内皮细胞中的表达。本初步报告为后续功能研究奠定了基础，以揭示它们在内皮细胞激活以及与包括肿瘤生长在内的脉管系统相关的病理生理事件中的作用。
