Regulation of Vascular Stabilization by Connexin 43

描述(申请人提供)：正常的血管由一小管内皮细胞与支持层的壁细胞(周细胞或平滑肌细胞)接触组成，该支撑层可稳定血管并使血管静止。相反，肿瘤形成的血管系统高度紊乱，表现出与壁细胞的减少和异常联系。肿瘤血管的这种缺陷给肿瘤控制带来了几个挑战：i)在正常组织中，壁细胞阻止内皮细胞的增殖，缺乏功能性的壁细胞联合使肿瘤能够刺激内皮细胞的增殖并产生新的血管来供给肿瘤；ii)缺乏壁细胞使血管更容易被肿瘤内皮细胞侵入，导致转移；iii)有缺陷的肿瘤血管的疏松减少了治疗药物的输送。我们试图通过治疗来理解克服这些缺陷。我们的初步数据表明，连接分子连接蛋白43(Cx43)在稳定正常血管中发挥着先前未知的作用。此外，乳腺和脑肿瘤细胞下调或失活壁细胞Cx43，以破坏它们与内皮细胞的相互作用。基于这些新的发现，我们将探讨一种假设，即肿瘤诱导的Cx43抑制在肿瘤血管不稳定中起主要作用，而血管Cx43的恢复将逆转这种有害影响。为了研究这一假说，我们提出了三个具体的目标：1)确定肿瘤暴露的血管细胞中Cx43活性的调节；2)确定改变的宿主Cx43是否影响同基因乳腺肿瘤的生长、血管生成或转移；以及3)确定改变的宿主Cx43是否影响血管的完整性和通透性。在目标1中，我们将使用药物抑制剂、特异性Cx43磷酸化突变体和缝隙连接缺陷突变体来确定肿瘤改变Cx43表达和功能的机制(S)。我们还将测试报告的化合物上调Cx43和/或增强血管稳定性的能力，以覆盖肿瘤诱导的血管细胞功能Cx43的丧失。在目标2和目标3中，我们将使用同基因小鼠乳腺肿瘤模型来研究在Cx43宿主缺失的背景下，肿瘤血管生成、转移、血管通透性和间质液体压力是否增加，以及Cx43过表达是否通过血管稳定来阻止或延迟这些事件。总之，这些研究将确定Cx43在血管稳定性中的作用，并阐明其在病理性血管系统中功能丧失的机制。这些研究的成功将使Cx43成为使肿瘤血管正常化的新靶点，从而抑制血管生成，潜在地减少转移并增强药物输送，最终导致更好的癌症控制。公共卫生相关性：在肿瘤中发现的漏水、不稳定的血管促进肿瘤生长，损害药物输送，并促进转移。拟议研究的成功将确定血管连接蛋白43是使肿瘤血管正常化的新靶点，从而抑制血管生成，减少转移，增强药物输送，最终导致更好的癌症控制。

DESCRIPTION (provided by applicant): A normal blood vessel is composed of a tubule of endothelial cells in contact with a supporting layer of mural cells (pericytes or smooth muscle cells) that stabilize the vessel and render the vessel quiescent. In contrast, the vasculature formed by a tumor is highly disorganized and exhibits decreased and abnormal association with mural cells. This defect in tumor vasculature causes several challenges in tumor control: i) while in normal tissues, mural cells prevent the endothelium from proliferating, lack of functional mural cell association allows tumor to stimulate endothelial proliferation and generate new vessels to feed the tumors; ii) the lack of mural cells makes vessels more susceptible to tumor intravasation, leading to metastasis; and iii) the leakiness of defective tumor vessels decreases delivery of therapeutic drugs. We seek to understand therapeutically overcome these defects. Our preliminary data demonstrate that the junctional molecule Connexin 43 (Cx43) plays a previously unidentified role in stabilization of normal blood vessels. Moreover, breast and brain tumor cells downregulate or inactivate mural cell Cx43 to disrupt their interaction with endothelial cell. Based on these novel findings we will investigate the hypothesis that tumor-induced inhibition of Cx43 plays a major role in destabilization of tumor blood vessels and that restoration of vascular Cx43 will reverse the deleterious effects. To investigate this hypothesis, we propose three specific aims: 1) To identify regulators of Cx43 activity in tumor-exposed vascular cells; 2) To determine whether altered host Cx43 affects the growth, angiogenesis, or metastasis of syngeneic mammary tumors; and 3) To determine whether altered host Cx43 affects vessel integrity and permeability. In Aim 1 we will use pharmacological inhibitors, site-specific Cx43 phosphorylation mutants, and gap junction-deficient Cx43 mutants to define the mechanism(s) by which tumor alters Cx43 expression and function. We will also test the ability of compounds reported to upregulate Cx43 and/or enhance vessel stability to override tumor-induced loss of functional Cx43 in vascular cells. In Aims 2 and 3, we will use a syngeneic mouse mammary tumor model to address whether tumor angiogenesis, metastasis, vascular permeability and interstitial fluid pressure are enhanced on a Cx43 host-deficient background, and whether Cx43 overexpression will prevent or delay these events via vascular stabilization. Together, these studies will define the role of Cx43 in blood vessel stability and elucidate the mechanism by which its function is lost in a pathological vasculature. Success of the proposed studies will identify Cx43 as a novel target to normalize tumor vasculature, thereby inhibiting angiogenesis, and potentially decreasing metastasis and enhancing drug delivery, ultimately leading to better cancer control. PUBLIC HEALTH RELEVANCE: The leaky, unstable blood vessels found in tumors facilitate tumor growth, impair drug delivery, and facilitate metastasis. Success of the proposed studies will identify vascular Connexin 43 as a novel target to normalize the tumor vasculature and thereby inhibit angiogenesis, decrease metastasis, and enhance drug delivery, ultimately leading to better cancer control.