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

描述(申请人提供)：在美国，每年有超过50万的自体静脉移植物被植入。然而，30%-60%的移植物在第一年内失败或出现临床上显著的狭窄，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认为新生内膜增生(NIH)，令人信服的证据表明，转化生长因子是导致早期失败的驱动因素。不幸的是，非选择性的封锁广泛 转化生长因子-β活性在减轻新生内膜增生形成方面取得了有限的成功，提示需要抑制特定的转化生长因子-β活性。决定转化生长因子受体特异性的一个主要机制是其I型受体Alk1或Alk5的激活。尽管Alk1在成熟的内皮细胞(ECs)和中膜平滑肌细胞(SMC)中的表达水平很低，但现有的证据表明，Alk1在静脉移植适应过程中在ECs和新生内膜SMC中被诱导表达。最近对血管生成和其他病理学的研究导致了一种新的理解，其中转化生长因子通过Alk1和Alk5发出信号来启动调节细胞生物学的相反作用。因此，我们假设血管壁对转化生长因子的反应依赖于内皮细胞和血管内皮细胞中Alk1和Alk5信号之间的平衡。对移植静脉壁的损伤使两种细胞类型的平衡倾向于Alk5信号，而Alk5信号反过来抑制内皮细胞的功能恢复，并维持SMC的炎症/合成表型，推动进展的NIH。选择性地阻断Alk5信号来恢复这种平衡将改善愈合反应并抑制NIH。为了验证这一假说，本项目旨在：1)通过验证的小鼠静脉移植模型和原代新生内膜SMC培养，确定SMC中Alk1和Alk5信号在调节新生内膜SMC表型和静脉移植物形态中的作用；2)评估ECs中竞争的Alk1和Alk5信号对新生EC单层的功能恢复、新SMC表型的调节和所导致的静脉移植物形态的影响；3)检测siRNA的治疗效果以及Alk1或Alk5信号的药物抑制在小鼠和人静脉移植物NIH发生中的作用。CreloxP系统将在成年小鼠的ECs或SMC中诱导Alk1或Alk5的选择性缺失，从而建立含有和不含EC或SMC特异性Alk1或Alk5的静脉移植物，以评估静脉移植物的形态、EC单层的修复以及新生内膜SMC的炎症表型。为了促进这些遗传途径产生的新知识的临床转化，特定的siRNA和新型药理抑制剂将被应用于抑制小鼠和体外人类静脉移植物中的Alk1和Alk5信号通路。然后将使用形态终点(例如NIH体积)和生物学终点(例如新生内膜细胞的表型属性)来评估这些方法的治疗效果。这些目标的完成不仅将为转化生长因子生物学的基本原理提供新的见解，还将产生新的策略来操纵复杂的生物过程，如静脉移植物壁适配。

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-ß is a driving factor for this early failure. Unfortunately, non-selective blockade of the broad TGF-ßactivities has yielded limited success in attenuating neointimal hyperplasia formation, suggesting inhibition of the specific TGF-ß activities is required. A primary mechanism that dictates TGF-ß 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-ß 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-ß 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-ß biology, but also generate novel strategies to manipulate complex biologic processes such as vein graft wall adaptation.