The Dichotomy of Alk1 and Alk5 Signaling Pathways in Vascular Response to Injury

Alk1 和 Alk5 信号通路在血管损伤反应中的二分法

• 批准号: 8465262
• 负责人: Zhihua Jiang
• 金额: $ 30.29万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8465262
• 关键词: Adult; Attenuated; Autologous; Automobile Driving; Biological Models; Biology; Blood Vessels; Bypass; CCL2 gene; Cell Culture Techniques; Cells; Cellular biology; Cessation of life; Clinical; Clinical Trials; Complex; Data; Development; Endothelial Cells; Endothelium; Equilibrium; Evaluation; Failure; Genes; Genetic; Genetic Recombination; Healed; Human; Hyperplasia; Implant; In Vitro; Inflammatory; Interleukin-6; Knowledge; Limb structure; Medial; Mediating; Mediator of activation protein; Modeling; Morphology; Mus; Myosin Heavy Chains; NF-kappa B; Nature; Organ; Outcome; Pathology; Patients; Performance; Pharmacologic Substance; Phenotype; Population; Process; Production; Property; Recovery of Function; Role; Secondary to; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Specificity; Stenosis; System; Tamoxifen; Testing; Therapeutic; Transforming Growth Factors; Translations; United States; Veins; Work; angiogenesis; cell type; clinical application; clinically relevant; clinically significant; cytokine; graft failure; graft healing; healing; improved; in vivo; inhibitor/antagonist; insight; leukemic stem cell; migration; monolayer; novel; novel strategies; programs; promoter; reagent testing; receptor; recombinase; repaired; response; response to injury; success; therapeutic effectiveness; tool; treatment strategy

DESCRIPTION (provided by applicant): Over a half million of autologous vein grafts are implanted annually in the United States. However, 30-60% of the grafts fails or develops a clinically significant stenosis within the first year, causing limb loss and death. The primary cause for early vein graft failure has been identified as neointimal hyperplasia (NIH) and compelling evidence has demonstrated that TGF-2 is a driving factor for this early failure. Unfortunately, non-selective blockade of the broad TGF-2 activities has yielded limited success in attenuating neointimal hyperplasia formation, suggesting inhibition of the specific TGF-2 activities is required. A primary mechanism that dictates TGF-2 specificity is the activation of its type I receptors Alk1 or Alk5. Although Alk1 is expressed at very low level in mature endothelium (ECs) and medial smooth muscle cells (SMCs), existing evidence suggests that Alk1 is induced in ECs and neointimal SMCs during vein graft adaptation. Recent studies for angiogenesis and other pathologies have led to an emergence of new understanding, wherein TGF-2 signals through Alk1 and Alk5 to initiate opposing effects on regulating cellular biology. We therefore hypothesize that the response of the vascular wall to TGF-2 relies on the balance between Alk1- and Alk5- signaling in both ECs and SMCs. Insult to the vein graft wall tips the balance in both cell types towards Alk5 signaling that in turn inhibits the functional recovery of ECs and upholds an inflammatory/synthetic phenotype for SMCs, driving progressive NIH. Selectively blocking Alk5 signaling to restore this balance will improve the healing response and inhibit NIH. To test this hypothesis, this project aims to: 1) Define the role of Alk1 and Alk5 signaling in SMCs in regulating the phenotype of neointimal SMCs and vein graft morphology via a validated murine vein graft model and primary neointimal SMC culture; 2) Evaluate the impact of the competing Alk1 and Alk5 signaling in ECs on functional recovery of the repopulated EC monolayer, modulation of neoSMC phenotype, and the resultant vein graft morphology; and 3) Examine the therapeutic effectiveness of siRNA and pharmaceutical inhibition of Alk1 or Alk5 signaling on the development of NIH in murine and human vein grafts. The CreloxP system will be utilized to induce selective deletion of Alk1 or Alk5 in ECs or SMCs in adult mice, so that vein grafts with and without EC or SMC specific Alk1 or Alk5 can be created for the evaluation of the vein graft morphology, the repair of the EC monolayer, and the inflammatory phenotype of neointimal SMCs. To facilitate the clinical translation of the new knowledge generated with these genetic approaches, specific siRNA and novel pharmacological inhibitors will be applied to inhibit Alk1 and Alk5 signaling pathways in both murine and ex vivo human vein grafts. The therapeutic effectiveness of these approaches will then be evaluated using both morphologic (e.g. NIH volume) and biologic (e.g. phenotypic properties of the neointimal cells) endpoints. Completion of these aims will not only provide new insights into the fundamentals of TGF-2 biology, but also generate novel strategies to manipulate complex biologic processes such as vein graft wall adaptation.

描述（由申请人提供）：美国每年植入超过 50 万个自体静脉移植物。然而，30-60% 的移植物在第一年内失败或出现临床上显着的狭窄，导致肢体丧失和死亡。早期静脉移植失败的主要原因已被确定为新生内膜增生 (NIH)，并且令人信服的证据表明 TGF-2 是这种早期失败的驱动因素。不幸的是，非选择性阻断广泛的 TGF-2 活性在减弱新内膜增生形成方面取得的成功有限，这表明需要抑制特定的 TGF-2 活性。决定 TGF-2 特异性的主要机制是激活其 I 型受体 Alk1 或 Alk5。尽管 Alk1 在成熟内皮细胞 (EC) 和内侧平滑肌细胞 (SMC) 中表达水平非常低，但现有证据表明 Alk1 在静脉移植适应过程中在 EC 和新生内膜 SMC 中被诱导。最近对血管生成和其他病理学的研究产生了新的认识，其中 TGF-2 通过 Alk1 和 Alk5 发出信号，启动对调节细胞生物学的相反作用。因此，我们假设血管壁对 TGF-2 的反应依赖于 EC 和 SMC 中 Alk1 和 Alk5 信号传导之间的平衡。对静脉移植壁的损害使两种细胞类型的平衡向 Alk5 信号传导倾斜，进而抑制 EC 的功能恢复并维持 SMC 的炎症/合成表型，从而导致进行性 NIH。选择性阻断 Alk5 信号传导以恢复这种平衡将改善愈合反应并抑制 NIH。为了验证这一假设，本项目旨在： 1) 通过经过验证的小鼠静脉移植模型和原代内膜 SMC 培养，明确 SMC 中 Alk1 和 Alk5 信号传导在调节新内膜 SMC 表型和静脉移植形态方面的作用； 2) 评估 EC 中竞争性 Alk1 和 Alk5 信号传导对重新填充的 EC 单层功能恢复、neoSMC 表型调节以及由此产生的静脉移植物形态的影响； 3) 检查 siRNA 和药物抑制 Alk1 或 Alk5 信号传导对小鼠和人静脉移植物中 NIH 发展的治疗效果。 CreloxP 系统将用于诱导成年小鼠 EC 或 SMC 中 Alk1 或 Alk5 的选择性删除，以便可以创建具有或不具有 EC 或 SMC 特异性 Alk1 或 Alk5 的静脉移植物，用于评估静脉移植物形态、修复EC 单层的变化和新内膜 SMC 的炎症表型。为了促进这些遗传方法产生的新知识的临床转化，将应用特异性 siRNA 和新型药理学抑制剂来抑制小鼠和离体人静脉移植物中的 Alk1 和 Alk5 信号通路。然后将使用形态学（例如 NIH 体积）和生物学（例如新内膜细胞的表型特性）终点来评估这些方法的治疗效果。这些目标的完成不仅将为 TGF-2 生物学的基础提供新的见解，而且还将产生操纵复杂生物过程（例如静脉移植壁适应）的新策略。