Role of Human Chymase in Smooth Muscle Contraction in Asthma

 描述(由申请人提供)：这是为加州大学旧金山分校(UCSF)医学系肺与重症监护科助理教授Aparna Sundaram博士重新提交的K08奖项的申请。桑达拉姆博士正在努力使自己成为哮喘领域的一名独立研究员。Sundaram博士和他的同事最近发表的数据表明，缺乏αvβ6整合素(调节转化生长因子-β活性)的小鼠可免受呼吸道过度活动的影响。此外，这种作用在一定程度上是通过在没有转化生长因子-β的情况下上调小鼠肥大细胞蛋白酶4(mMCP-4)来实现的。Sundaram博士发现mMCP-4抑制IL-13增强小鼠气管环的收缩。人类凝乳酶是mMCP-4最接近的同源基因，桑达拉姆博士已经证明，凝乳酶也能保护机体免受IL-4的侵袭。 13增强小鼠和人的气道环的收缩。糜酶不会通过影响肌动蛋白-肌球蛋白的交叉桥接来调节收缩，所以桑达拉姆博士把重点放在了假设上，即它影响了收缩装置与潜在的细胞外基质之间的联系。为了支持这一假说，桑达拉姆博士已经证明，凝乳酶可以裂解纤维连接蛋白，并会削弱整合素介导人肌细胞与纤维连接蛋白的黏附，但不会影响胶原蛋白或玻璃体连接蛋白。此外，用RGD肽(在纤维连接蛋白上发现的一种整合素结合序列)处理气管环会削弱IL-13增强的收缩，联合使用RGD肽和糜酶不能比单独使用RGD肽或糜酶提供进一步的保护。 在这项提案中，Sundaram博士将系统地研究糜酶 调节收缩反应。她将探索凝乳酶对整合素表达的影响，确定哪些特定的整合素(S)在纤维连接蛋白介导的粘连中起重要作用，研究整合素的丢失对局部粘连的放大、分布和信号的影响，以及对体外收缩反应和体内过敏原攻击反应的影响(目标1)。她还将进一步探索过敏原挑战和细胞因子暴露在揭示糜酶保护作用方面的作用。她将通过研究过敏原挑战对糜酶介导的细胞外基质保护和组织的影响来做到这一点。她还将研究各种细胞因子对整合素表达的影响，以及它们对黏附、焦点黏附复合体信号和糜酶介导的保护的贡献(目标2)。 围绕这些目标组织，并在正式指导和课程作业的指导下，Sundaram博士 将追求以下目标：(1)了解人类乳糜酶对细胞因子诱导的呼吸道过度活动的保护机制；(2)发展技能、专业知识和填补知识空白，使之能够平稳过渡到科学独立。Sundaram博士组建了由加州大学旧金山分校教职员工组成的多元化、多学科的指导团队，成员包括主要导师、医学教授和国际公认的整合素生物学专家Dean Seppard，共同导师、医学教授和多产的肥大细胞生物学基础科学研究员George Ceh，以及委员会成员，包括研究哮喘细胞免疫反应的解剖学助理教授Chris Allen，医学教授和肺生物中心副主任及胸膜疾病国际专家Courtney Broaddus，以及研究与哮喘和肉芽肿性肺部疾病相关的翻译研究的医学副教授Laura Koth。Sundaram博士的研究提案侧重于Chymase的新角色，再加上她的结构化训练计划，将使她能够开发出必要的 R01拨款申请的技能和初步数据，以及建立有别于 她的导师允许她成为一名独立的内科科学家。

 DESCRIPTION (provided by applicant): This is a resubmission application for a K08 award for Dr. Aparna Sundaram, an Assistant Professor in the Division of Pulmonary & Critical Care, Department of Medicine, at the University of California, San Francisco (UCSF). Dr. Sundaram is working to establish herself as an independent investigator in the field of asthma. Recent data published by Dr. Sundaram and colleagues has suggested that mice lacking the αvβ6 integrin, which modulates TGF-β activity, are protected from airway hyperactivity. Furthermore, this effect is mediated in part by up-regulation of mouse mast cell protease 4 (mMCP-4) in the absence of TGF-β. Dr. Sundaram found that mMCP-4 inhibited IL-13 enhanced contraction in mouse tracheal rings. Human chymase is the closest orthologue of mMCP-4, and Dr. Sundaram has shown that chymase is also protective against IL- 13 enhanced contraction in both mouse and human airway rings. Chymase does not modulate contraction by affecting actin-myosin cross-bridging, so Dr. Sundaram has focused on the hypothesis that it affects linkage of the contractile apparatus to the underlying extracellular matrix. In support of this hypothesis, Dr. Sundaram has shown that chymase cleaves fibronectin, and impairs integrin-mediated adhesion of smooth muscle cells to fibronectin, but not collagen or vitronectin. Furthermore, treatment of tracheal rings with an RGD peptide (an integrin-binding sequence found on fibronectin) impairs IL-13 enhanced contraction, and combined treatment with RGD peptide and chymase does not confer further protection over either RGD peptide or chymase alone. In this proposal Dr. Sundaram will systematically investigate the mechanism by which chymase modulates contractile responses. She will explore the effect of chymase on integrin expression, determine which specific integrin(s) are important in fibronectin-mediated adhesion, investigate the effect of loss of integrin's on enlargement, distribution, and signaling within focal adhesions as well as effects on ex vivo responses to contraction and in vivo responses to allergen challenge (Aim 1). She will also further explore the role of allergen challenge and cytokine exposure on unmasking the protective effect of chymase. She will do this by investigating the effect of allergen challenge on chymase-mediated protection and organization of the extracellular matrix. She will also study the effect of various cytokines on integrin expression and their contribution to adhesion, focal adhesion complex signaling, and chymase-mediated protection (Aim 2). Organized around these aims and guided by both formal mentorship and coursework, Dr. Sundaram will pursue the following goals: (1) to understand the mechanism of protection against cytokine-induced airway hyperactivity by human chymase; and (2) to develop skills, expertise, and fill knowledge gaps to enable a smooth transition to scientific independence. Dr. Sundaram has assembled a diverse and multidisciplinary mentoring team of UCSF faculty including primary mentor Dean Sheppard, Professor of Medicine and an internationally recognized expert in integrin biology, co-mentor George Caughey, Professor of Medicine and a prolific basic science researcher in mast cell biology, and committee members including Chris Allen, Assistant Professor of Anatomy with research interests in cellular immune responses in asthma, Courtney Broaddus, Professor of Medicine and the Associate Director of the Lung Biology Center as well as international expert in pleural diseases, and Laura Koth, Associate Professor of Medicine whose research encompasses translational studies relating to asthma and granulomatous lung diseases. Dr. Sundaram's research proposal focusing on the novel role of chymase coupled with her structured training plan will allow her to develop the necessary skills and preliminary data for an R01 grant application as well as establish a niche distinct from her mentors to allow her to become an independent physician-scientist.