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

描述(申请人提供)：腹主动脉瘤(AAA)，其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消退现有的动脉瘤是困难的，因为成年细胞合成弹性蛋白很差，而且没有可用的工具来诱导忠实的弹性蛋白再生。因此，我们的长期目标是研究在AAA内实现弹性蛋白再生的策略，以延迟或取消手术干预。我们确定，基于基质糖胺聚糖透明质酸(HA)四聚体和转化生长因子-2的弹力生成信号协同上调健康成人血管平滑肌细胞的弹性蛋白基质的合成和组装，并在较小程度上上调动脉瘤细胞的合成和组装。这一结果预示着这些信号对AAS内类似的弹性蛋白再生具有巨大的实用性。然而，可溶性弹性蛋白前体的募集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信号(LOX，一种弹性蛋白交联酶和Cu2+)，与弹性信号同时提供或独立提供，对培养的健康和动脉瘤成年大鼠主动脉SMC(RASMCs)的弹性蛋白合成、基质组装和细胞表型(例如，弹性蛋白酶和基质金属蛋白酶的释放)的影响。我们还试图研究优化的线索对诱导的大鼠AAA中弹性蛋白基质再生的有效性。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性模型，在该模型中，RASMC(健康的和动脉瘤的)将被提供合成、组装和交联原弹性蛋白前体的线索。在外源性模型中，SMC(健康的和动脉瘤的)将只提供LOX和Cu2信号来上调外源性原弹性蛋白的细胞组装和成熟。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)提供的铜离子在优化LOX信号的同时，对RASMC培养中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，Aim 3将测试优化的交联提示(LOX、Cu2)用于弹性蛋白基质的原位细胞组装的有效性，以稳定处于不同发育阶段的诱发大鼠主动脉瘤(AA)，当原弹性蛋白由弹性提示内源性提示时，或(B)外源性供应时。我们预计该项目成果将为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，可以作为一种独立的选择，或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有关的应用，包括增强组织工程构建中的弹性蛋白合成、组装和基质质量，恢复去弹性的同种异体血管和异种血管中的弹性蛋白稳态，甚至可能用作研究早期形态形成过程中的弹性发生和成年血管伤口愈合的体外模型。与公共卫生相关：腹主动脉瘤(AAA)是一种潜在的致命性疾病，困扰着主要血管，其特征是血管壁灵活性丧失，最终结构减弱和破裂。这是由于橡胶状蛋白纤维(弹性蛋白)的分解和丢失造成的，弹性蛋白通常帮助血管在变形后恢复其形状和形状。由于血管内的细胞本身不能产生新的弹性蛋白，本研究建议在培养的或活的血管内提供细胞，生物分子的组合将诱导细胞(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(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开发新的非手术治疗策略，通过诱使体内细胞再生新的弹性蛋白结构或修复和稳定现有的AAA，从而阻止AAA的进展，甚至使其退化。

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.