Role of Src family kinases in endothelial cell biology

描述(申请人提供)：脑内皮屏障的特征是具有低细胞旁通透性的特殊内皮细胞。与基底膜和其他类型的细胞一样，血脑屏障(BBB)是正常脑代谢的重要调节因素。血管内皮生长因子是一种由肿瘤分泌的生长因子，可诱导血管通透性(VP)，破坏血脑屏障的完整性。我们以前的研究已经证明，对于血管内皮生长因子诱导的血管通透性(VP)，宿主间隔中需要Src，特别是在肺和脑中。我们利用抗渗漏的src基因敲除小鼠的表型来检测肿瘤生长和转移的特征。这些研究表明，缺少Src会导致血管内皮生长因子诱导的血管VP减少，从而保护血管免受肺转移的影响。在大脑中，血管内皮生长因子诱导的VP的减少也与Src介导的FAK磷酸化的变化有关。最近的数据表明，与周围细胞类型相比，FAK在脑血管中丰富，是Src介导的VP的靶点。我们假设FAK是维持血脑屏障完整性的关键中间体。在目标1中，我们将表征Src在调节血管内皮生长因子诱导的FAK激活和脑血管VP中的作用。这些研究将检验VP表型的分子基础，并将包括BMK1和eNOS的特征。在目标2中，我们将确定Src是否调节血管内皮生长因子刺激的脑血管中的BMK1。在目标3中，我们将研究eNOS在src-vs.BMK1介导的血管内皮细胞生长因子诱导的VP中的作用。虽然这些研究将为血脑屏障的调节提供新的和重要的见解，但研究血脑屏障在肿瘤转移中的作用的研究相对较少。因此，我们将利用动物模型来确定FAK、BMK1和eNOS在乳腺肿瘤脑转移过程中的作用。这些研究将建立在我们之前对Src在宿主间隔室中的作用的研究的基础上，并为表征颅内肿瘤的生长和侵袭提供比较。一组人脑星形细胞瘤可用于人脑胶质瘤生长和渗透的异种小鼠模型。这些研究的结果将在不同恶性程度的人脑肿瘤样本中得到验证，以确定VP和BBB在肿瘤生长和侵袭的不同阶段的临床病理相关性。

DESCRIPTION (provided by applicant): The endothelial barrier of the brain is characterized by specialized endothelial cells with low paracellular permeability. Along with the basement membrane and other cell types, the blood brain barrier (BBB) is an important regulator of normal brain metabolism. VEGF is a growth factor secreted by tumors that can induce vascular permeability (VP) and disrupt the integrity of the BBB. Our previous studies have demonstrated a Src requirement in the host compartment for VEGF-induced vascular permeability (VP), particularly in the lung and brain. We have exploited the 'leakage-resistant1 phenotype of Src-knockout mice to examine the characteristics of tumor growth and metastasis. These studies have revealed that an absence of Src leads to reduced VEGF-induced VP of blood vessels providing protection from lung metastases. In the brain, a reduction in VEGF-induced VP has also been associated with Src-mediated changes in FAK phosphorylation. Recent data indicate that FAK is enriched in brain blood vessels compared to surrounding cell types, and is a target of Src-mediated VP. We hypothesize that FAK is a key intermediate in the maintenance of the integrity of the BBB. In Aim 1 we will characterize the role of Src in regulating VEGF- induced FAK activation and VP of brain blood vessels. These studies will examine the molecular basis for the VP phenotype and will include the characterization of BMK1 and eNOS. In Aim 2 we will determine whether Src regulates BMK1 in VEGF-stimulated brain blood vessels. In Aim 3 we will examine the role of eNOS in Src- vs. BMK1-mediated VEGF-induced VP. While these studies will provide novel and important insights into the regulation of the BBB, relatively few studies have examined the role of the BBB in tumor metastasis. Therefore, we will use animal models to determine the function of FAK, BMK1 and eNOS in the host compartment during breast tumor metastasis to the brain. These studies will build upon our previous studies of the role of Src in the host compartment in Src-mediated metastases to the lung and provide a comparison for the characterization of the growth and invasion of intracranial tumors. A panel of human astrocytomas is available for xenograft mouse models of human glioma growth and infiltration. The results of these studies will be validated in human brain tumor samples of varying malignancy to determine the clinicopathological relevance of VP and the BBB in various stages of tumor growth and invasion.