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）的特征是主动脉弹性蛋白基质降解，导致血管弱化和破裂，每年在美国导致15，000例死亡，主要发生在老年人和患有遗传性基质疾病的人中。通过恢复健康的弹性蛋白结构来使现有的动脉瘤退化是困难的，因为成年细胞合成弹性蛋白的能力很差，并且没有工具可用于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内的弹性蛋白再生的策略，以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的四聚体的弹性蛋白生成线索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平滑肌细胞。这些结果预示着这些线索对AA内类似弹性蛋白再生的巨大效用。然而，招募和交联的可溶性弹性蛋白前体到一个稳定的矩阵是低效的，必须上调，不足，我们建议解决。因此，我们的目的是研究与弹性蛋白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Cu 2+离子）对弹性蛋白合成、基质组装和细胞表型（例如，弹性蛋白酶和MMP释放）。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“内源性模型”，其中RASMC（健康和不对称的）将被提供线索以合成、组装和交联原弹性蛋白前体。在外源性模型中，SMC（健康的和不典型的）将被提供LOX和Cu 2+线索，仅用于上调外源性弹性蛋白原的细胞组装和成熟。目的1将研究剂量特异性的好处外源性，脂氧合酶弹性蛋白的合成，基质组装，和细胞表型，RASMC培养。目的2将评估从铜纳米颗粒（CuNP）递送的铜离子与优化的LOX线索同时对LOX活性和对RASMC培养物内的弹性蛋白合成和基质组装以及细胞表型的剂量特异性影响。最后，目的3将测试优化的交联因子（LOX，Cu 2+）用于弹性蛋白基质的原位细胞组装的效用，以稳定处于不同发育阶段的诱导的大鼠主动脉瘤（AA），当弹性蛋白原（A）由弹性蛋白生成因子内源性地促进，或（B）外源性地提供时。我们预计项目结果将为AAA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，可作为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物中的弹性蛋白合成、组装和基质质量，恢复去弹性化血管同种异体移植物和异种移植物中的弹性蛋白稳态，甚至可能用作体外模型来研究早期形态发生期间的弹性蛋白生成和成人血管中的伤口愈合。公共卫生相关性：腹主动脉瘤（AAA）是影响主要血管的潜在致命性疾病，其特征在于血管壁柔韧性的丧失及其最终的结构弱化和破裂。这是由于橡胶样蛋白质纤维（弹性蛋白）的分解和损失而发生的，这些纤维通常有助于血管在变形后恢复其形状和形式。由于血管内的细胞本身不能产生新的弹性蛋白，因此本研究提出在培养物中或在活血管内提供细胞，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）仅组装也同时提供给它们的弹性蛋白前体。该项目的成果可以显着有利于开发新的非手术治疗策略，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.