Collagen I, Hyaluronan, and Aging

描述（由申请人提供）：人们普遍认为透明质酸（HA）是一种细胞外基质（ECM）糖胺聚糖，是炎症、血管生成和伤口修复的主要参与者。值得注意的是，HA（二糖 D-葡萄糖醛酸/D-N-乙酰葡萄糖胺的简单聚合物）对血管生成具有与其链长度相关的不同影响。具体而言，通常具有高分子量 (HMW-HA)（例如，超过 1 x 103 kDa）的天然 HA 抑制血管生成，而低分子量 (LMW-HA)（例如，小于 3 kDa/小于 12 个寡聚物）的 HA 则刺激血管生成。然而，人们对衰老如何影响 HA 以及这种影响是否会导致与衰老相关的愈合缺陷知之甚少。更好地了解这一领域可能会导致开发出改进的老年人伤口修复疗法。在这种临床潜力的背景下，我们最近发现，从老年小鼠的结缔组织（肌腱）中提取的 ECM 提取物中的 HA 含量明显高于从年轻小鼠中提取的 HA。此外，我们注意到，老年小鼠的真皮切除伤口（其愈合速度比年轻小鼠的伤口慢得多）和从老年人类供体培养的成纤维细胞比年轻小鼠产生更多的 HA。这些发现强调需要进行具有以下目标的进一步研究：（1）定量衰老对伤口组织中HA浓度和分子量的影响，（2）确定导致观察到的HA浓度和分子量变化的细胞介导过程（例如HA合成/降解），以及（3）确定HA浓度和/或分子量的变化是否以及如何影响血管生成，血管生成是伤口修复的关键过程，已知在老年宿主中存在缺陷。 该提案将追求以下 2 个假设：1) ECM 中 HA 与年龄相关的增加伴随着 HA 分子量分布的变化，这不利于血管生成，但当受到刺激时，2) 来自老年供体的内皮细胞能够对 HA 的促血管生成形式做出反应。以下 2 个具体目标将解决这 2 个假设：（目标 1）确定从年轻、中年和老年小鼠中分离的真皮切除伤口组织和培养的伤口成纤维细胞中的 HA 浓度和分子量分布，以及（目标 2）确定 HA 分子量类别（高、中和低）对体外血管生成的影响。对于体外研究，我们将利用 3D 胶原蛋白中血管生成的互补模型：小鼠微血管外植体的发芽和从不同年龄的供体中分离的人微血管内皮细胞 (hmEC) 的管状发生。两者都是先前未经检验的老化过程中HA和内皮细胞功能的模型。 这些目标和相关的具体目标将有助于我们的长期目标，即更好地了解 ECM 中 HA 与年龄相关的变化，以便检查利用内源性和外源性 HA 在体内组织修复过程中调节血管生成的效果。 公共健康相关性：我们认为透明质酸 (HA)——二糖 D-葡萄糖醛酸/D-N-乙酰葡萄糖胺的简单聚合物——是细胞外基质的重要组成部分，在衰老组织修复过程中调节血管生成。在本提案中，我们将使用 2 个相关但独立的具体目标，利用动物和人类衰老模型来测试以下假设：1）ECM 中 HA 与年龄相关的增加伴随着 HA 分子量分布的变化，这不利于血管生成，但当受到刺激时，2）来自老年供体的内皮细胞能够对 HA 的促血管生成形式做出反应。更好地了解细胞外基质中 HA 与年龄相关的变化，将有助于研究利用内源性和外源性 HA 在体内组织修复过程中调节血管生成的效果。

DESCRIPTION (provided by applicant): It is generally accepted that hyaluronan (HA), an extracellular matrix (ECM) glycosaminoglycan, is a major participant in inflammation, angiogenesis, and wound repair. Notably, HA - a simple polymer of the disaccharide D-glucuronic acid/D-N-acetylglucosamine - has differential effects on angiogenesis that are related to its chain length. Specifically, native HA, often of high molecular weight (HMW-HA) (e.g., over 1 x 103 kDa) is inhibitory to angiogenesis, whereas HA of low molecular weight (LMW-HA) (e.g., less than 3 kDa/less than 12 oligomers) stimulates angiogenesis. However, little is known about how aging affects HA and whether such effects might contribute to deficiencies in healing that are associated with aging. A better understanding of this area may lead to the development of improved therapies for wound repair in the aged. In the context of this clinical potential, we have recently found that ECM extracts from connective tissue (tendons) of aged mice contain significantly greater amounts of HA than that obtained from young mice. Moreover, we noted that dermal excisional wounds of aged mice (which heal much more slowly than wounds of young mice) and fibroblasts cultured from aged human donors produce significantly more HA than their young counterparts. These findings underscore the need for further studies that have the following objectives: (1) quantitate the influence of aging on HA concentration and molecular weight in wound tissue, (2) identify cell-mediated processes (e.g., HA synthesis/degradation) responsible for any observed changes in HA concentration and molecular weight, and (3) determine whether and how changes in HA concentration and/or molecular weight influence angiogenesis, a process critical to wound repair that is known to be deficient in aged hosts. This proposal will pursue the following 2 hypotheses: 1) age-associated increases in HA in ECM are accompanied by changes in HA molecular weight profiles that are detrimental to angiogenesis, but when stimulated, 2) endothelial cells from older donors are able to respond to pro-angiogenic forms of HA. The following 2 Specific Aims will address these 2 hypotheses: (Aim 1) determine HA concentration and molecular weight profiles in dermal excisional wound tissue and cultured wound fibroblasts isolated from young, middle aged, and aged mice and (Aim 2) determine the influence of HA molecular weight classes (high, medium, and low) on angiogenesis in vitro. For the in vitro studies, we will utilize complementary models of angiogenesis in 3D collagen: sprouting of murine microvessel explants and tubulogenesis of human microvascular endothelial cells (hmECs) isolated from donors of a range of ages. Both are previously unexamined models of HA and endothelial cell function in aging. These objectives and the associated specific aims will contribute to our long-term goal, which is to better understand age-related changes in HA in the ECM in order to examine the effect of utilizing endogenous and exogenous HA to modulate angiogenesis during tissue repair in vivo. PUBLIC HEALTH RELEVANCE: We propose that hyaluronan (HA)- a simple polymer of the disaccharide D-glucuronic acid/D-N- acetylglucosamine - is an important component of the extracellular matrix that regulates angiogenesis during tissue repair in aging. In this proposal we will use 2 related, but independent, specific aims that employ both animal and human models of aging to test the hypotheses that: 1) age-associated increases in HA in ECM are accompanied by changes in HA molecular weight profiles that are detrimental to angiogenesis, but when stimulated, 2) endothelial cells from older donors are able to respond to pro-angiogenic forms of HA. A better understanding of aged-related changes in HA in the extracellular matrix will allow for studies that exam the effect of utilizing endogenous and exogenous HA to modulate angiogenesis during tissue repair in vivo.