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 (AAA) 的特点是主动脉弹性蛋白基质降解并由此导致血管衰弱和破裂，在美国每年导致 15,000 人死亡，主要是老年人和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消退现有的动脉瘤是困难的，因为成体细胞合成弹性蛋白的能力较差，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使 AAA 内弹性蛋白再生的策略，从而延迟或消除手术干预。我们确定，基于透明质酸 (HA) 四聚体（一种基质糖胺聚糖）和 TGF-2 的弹性生成线索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对 AA 内类似的弹性蛋白再生具有巨大的效用。然而，将可溶性弹性蛋白前体招募和交联成稳定基质的效率低下，必须上调，这是我们建议解决的不足。因此，我们的目标是研究与弹性生成线索同时或独立提供的此类线索（LOX、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 SMC (RASMC) 的弹性蛋白合成、基质组装和细胞表型（例如弹性蛋白酶和 MMP 释放）的影响。我们还试图研究诱导大鼠 AAA 中弹性蛋白基质再生的优化线索的功效。在提出的三个目标中，我们将同时研究弹性蛋白基质再生的“内源模型”，其中将为 RASMC（健康和动脉瘤）提供合成、组装和交联原弹性蛋白前体的线索。在外源模型中，向 SMC（健康的和动脉瘤的）提供 LOX 和 Cu2 信号，仅用于上调外源弹性蛋白原的细胞组装和成熟。目标 1 将研究 RASMC 培养物中外源性 LOX 对弹性蛋白合成、基质组装和细胞表型的剂量特异性益处。目标 2 将评估铜纳米颗粒 (CuNP) 传递的铜离子与优化的 LOX 线索同时对 RASMC 培养物中 LOX 活性、弹性蛋白合成、基质组装和细胞表型的剂量特异性影响。最后，目标 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，以稳定各个发育阶段的诱导大鼠主动脉瘤（AA），此时原弹性蛋白是（A）由弹性线索内源性促进，或（B）外源性供应。我们预计该项目的成果将为 AAA 提供更有效的治疗选择，基于弹性蛋白基质的原位再生和稳定，可以作为独立的选择或与现有的手术或未来的药理学方法结合使用。该项目成果的其他应用，特别是与健康血管细胞研究有关的应用，包括增强组织工程结构中的弹性蛋白合成、组装和基质质量，恢复去弹性血管同种异体移植物和异种移植物中的弹性蛋白稳态，甚至可能作为体外模型来研究早期形态发生期间的弹性生成和成体血管的伤口愈合。公众健康相关性：腹主动脉瘤 (AAA) 是一种影响主要血管的潜在致命性疾病，其特点是血管壁灵活性丧失，最终导致结构减弱和破裂。这是由于橡胶状蛋白纤维（弹性蛋白）的分解和损失而发生的，而橡胶状蛋白纤维通常有助于血管在变形后恢复其形状和形态。由于血管内的细胞本身不能产生新的弹性蛋白，因此本研究建议为培养物或活体血管内的细胞提供生物分子的组合，这些生物分子将诱导细胞（a）合成新的可溶性弹性蛋白构件（前体）并进一步将它们组装成纤维结构，或（b）仅组装同时提供给它们的弹性蛋白前体。该项目的成果可以极大地有利于新的非手术治疗策略的开发，这些策略可以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来阻止现有 AAA 的进展，甚至使现有的 AAA 退化。

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.