Cues for cell-mediated regeneration of elastin matrix to stabilize aortic aneurys

描述（由申请人提供）：腹主动脉瘤（AAAs）的特征是主动脉弹性蛋白基质降解，导致血管变弱和破裂，在美国每年导致15,000人死亡，主要是老年人和那些患有遗传性基质疾病的人。通过恢复健康的弹性蛋白结构来恢复现有动脉瘤是困难的，因为成人细胞合成弹性蛋白的能力很差，而且没有工具可以诱导忠实的弹性蛋白再生。因此，我们的长期目标是研究策略，使弹性蛋白再生在AAAs，从而延迟或消除手术干预。我们确定，基于透明质酸（HA）、基质糖胺聚糖和TGF-2的四聚体的弹性提示可协同上调健康成人血管平滑肌细胞的弹性蛋白基质合成和组装，并在较小程度上上调动脉瘤细胞的弹性蛋白基质合成和组装。结果预示着这些线索在AAs内类似弹性蛋白再生中的巨大效用。然而，可溶性弹性蛋白前体的招募和交联进入稳定的基质是低效的，必须上调，这是我们建议解决的不足。因此，我们的目的是研究这些线索（LOX，一种弹性蛋白交联酶和Cu2+离子）对培养的健康和动脉瘤成年大鼠主动脉SMCs （RASMCs）的弹性蛋白合成、基质组装和细胞表型（如弹性酶和MMP释放）的影响，这些线索与弹性蛋白交联酶同时提供或独立于弹性源性线索。我们还试图研究优化的线索对诱导大鼠AAAs中弹性蛋白基质再生的功效。在这三个目标中，我们将并行研究弹性蛋白基质再生的“内源性模型”，其中RASMCs（健康和动脉瘤）将提供合成、组装和交联对弹性蛋白前体的线索。在外源性模型中，SMCs（健康的和动脉瘤的）将只提供LOX和Cu2+线索来上调外源性tropoelastin的细胞组装和成熟。目的1将研究外源性LOX对RASMC培养中弹性蛋白合成、基质组装和细胞表型的剂量特异性益处。目的2将评估铜纳米颗粒（CuNP）铜离子递送的剂量特异性效应，同时优化LOX线索，对RASMC培养中LOX活性、弹性蛋白合成、基质组装和细胞表型的影响。最后，目的3将测试优化的交联线索（LOX, Cu2+）在弹性蛋白基质原位细胞组装中的效用，以稳定诱导的大鼠主动脉瘤（AAs）在不同发育阶段，当弹力蛋白是(A)由弹性源线索内源性提示或(B)外源性供应时。我们期望项目的结果能够为AAAs提供更有效的治疗选择，基于原位再生和弹性蛋白基质的稳定，这可以作为一个单独的选择，也可以与现有的手术或未来的药理学方法相结合。该项目成果的其他应用，特别是与健康血管细胞研究相关的应用，包括增强组织工程构建中弹性蛋白的合成、组装和基质质量，在去弹性的异体和异种血管移植中恢复弹性蛋白的稳态，甚至可能作为研究早期形态发生和成人血管伤口愈合过程中的弹性发生的体外模型。公共卫生相关性：腹主动脉瘤（AAAs）是影响大血管的潜在致命疾病，其特征是血管壁失去灵活性，最终结构减弱和破裂。这是由于通常帮助血管在变形后恢复其形状和形状的橡胶样蛋白质纤维（弹性蛋白）的分解和损失。由于血管内的细胞本身不能产生新的弹性蛋白，本研究建议为培养细胞或活血管内的细胞提供一种生物分子的组合，这种组合可以诱导细胞(a)合成新的可溶性弹性蛋白构建块（前体）并进一步将其组装成纤维结构，或者(b)仅组装同时提供给它们的弹性蛋白前体。该项目的成果可以显著促进新的非手术治疗策略的发展，通过诱导细胞再生新的弹性蛋白结构或修复和稳定现有的弹性蛋白结构，可以阻止现有AAAs的进展甚至倒退。

DESCRIPTION (provided by applicant): Abdominal aortic aneurysms (AAAs), characterized by degrading aortic elastin matrix and resultant vessel weakening and rupture, causes 15,000 deaths in the United States anually, primarily amongst seniors, and those suffering from inherited matrix disorders. Regression of existing aneurysms by restoring healthy elastin architecture is difficult since adult cells poorly synthesize elastin and no tools are available to induce faithful elastin regeneration. Thus, our long-term goal is to investigate strategies to enable elastin regeneration within AAAs, so as to delay or eliminate surgical intervention. We determined that elastogenic cues based on tetramers of hyaluronan (HA), a matrix glycosaminoglycan, and TGF-2 synergestically upregulate elastin matrix synthesis and assembly by healthy adult vascular smooth muscle cells, and to a lesser extent by aneurysmal cells. The outcomes portend tremendous utility of these cues to similar elastin regeneration within AAs. However, recruitment and crosslinking of soluble elastin precursors into a stable matrix is inefficient and must be up-regulated, an insufficiency we propose to address. Our objective is thus to investigate impact of such cues (LOX, an elastin crosslinking enzyme, and Cu2+ ions), provided concurrent to or independent of elastogenic cues, on elastin synthesis, matrix assembly, and cell phenotype (e.g., elastase and MMP release) by cultured healthy and aneurysmal adult rat aortic SMCs (RASMCs). We also seek to investigate the efficacy of the optimized cues for elastin matrix regeneration in induced rat AAAs. In each of three proposed aims, we will in parallel investigate an `endogenous model' of elastin matrix regeneration, wherein RASMCs (healthy and aneurysmal) will be provided cues to both synthesize, and assemble and crosslink tropoelastin precursors. In the exogenous model, SMCs (healthy and aneurysmal) will be provided LOX and Cu2+ cues only to upregulate cellular assembly and maturation of exogenous tropoelastin. Aim 1 will investigate dose-specific benefits of exogenous, LOX to elastin synthesis, matrix assembly, and cell phenotype, within RASMC cultures. Aim 2 will evaluate dose-specific effects of copper ion delivery from copper nanoparticles (CuNP), concurrent with optimized LOX cues, to LOX activity and to elastin synthesis and matrix assembly and cell phenotype within RASMC cultures. Finally, aim 3 will test utility of optimized crosslinking cues (LOX, Cu2+) for in situ cellular assembly of elastin matrices, to stabilize induced rat aortic aneurysms (AAs) in various stages of development, when tropoelastin is (A) endogenously prompted by elastogenic cues, or (B) exogenously supplied. We expect the project outcomes to offer more effective treatment options for AAAs, based on both in situ regeneration and stabilization of elastin matrices that may be employed as a stand-alone option or in consort with existing surgical or future pharmacological approaches. Other applications of the project outcomes, specifically those pertaining to studies of healthy vascular cells, include augumenting elastin synthesis, assembly, and matrix quality within tissue engineered constructs, restoring elastin homeostasis in de-elasticized vascular allografts and xenografts, and possibly even serving as in vitro models to investigate elastogenesis during early morphogenesis, and wound healing in adult vessels. PUBLIC HEALTH RELEVANCE: Abdominal aortic aneurysms (AAAs) are potentially fatal conditions afflicting major blood vessels, which are characterized by a loss of blood vessel wall flexibility, and their ultimate structural weakening and rupture. This occurs due to breakdown and loss of rubber-like protein fibers (elastin) that normally help vessels restore their shape and form after deformation. Since cells within blood vessels cannot themselves produce new elastin, this study proposes to provide cells in culture, or within living blood vessels, a combination of biological molecules that will either induce cells to (a) synthesize new soluble elastin building blocks (precursors) and further assemble them into fiber structures, or (b) only assemble elastin precursors that are also simultaneously provided to them. The project outcomes can significantly benefit the development of new, non-surgical treatment strategies that can halt progress of or even regress existing AAAs by coaxing cells within to regenerate new elastin structures or repair and stabilize existing ones.