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

描述（由申请人提供）：在美国，每年有超过50万例自体静脉移植物植入。然而，30-60%的移植物在第一年内失败或发展为临床显著狭窄，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），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，广泛的TGF-2活性的非选择性阻断在减弱新生内膜增生形成方面取得了有限的成功，这表明需要抑制特异性TGF-2活性。决定TGF-2特异性的主要机制是其I型受体Alk 1或Alk 5的活化。尽管Alk 1在成熟内皮细胞（EC）和中膜平滑肌细胞（SMC）中表达水平很低，但现有证据表明，在静脉移植物适应过程中，Alk 1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已经导致了新的理解的出现，其中TGF-2通过Alk 1和Alk 5发出信号，以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Alk 1-和Alk 5-信号传导之间的平衡。对静脉移植物壁的损伤使两种细胞类型的平衡向Alk 5信号传导倾斜，Alk 5信号传导反过来抑制EC的功能恢复，并维持SMC的炎症/合成表型，从而推动NIH的进展。选择性阻断Alk 5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，本项目的目的是：1）通过有效的小鼠静脉移植模型和原代新生内膜SMC培养，确定SMC中Alk 1和Alk 5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EC中竞争性Alk 1和Alk 5信号传导对再增殖EC单层的功能恢复、新SMC表型的调节的影响，以及所得到的静脉移植物形态;以及3）检查siRNA和Alk 1或Alk 5信号传导的药物抑制对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Alk 1或Alk 5的选择性缺失，从而可以创建具有和不具有EC或SMC特异性Alk 1或Alk 5的静脉移植物，用于评价静脉移植物形态、EC单层的修复和新生内膜SMC的炎性表型。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Alk 1和Alk 5信号通路。然后将使用形态学（例如NIH体积）和生物学（例如新生内膜细胞的表型特性）终点评价这些方法的治疗有效性。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（如静脉移植物壁适应）的新策略。

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.