Collagen I, Hyaluronan, and Aging

描述(申请人提供)：透明质酸(HA)，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--D-葡萄糖醛酸/D-N-乙酰氨基葡萄糖的简单聚合物--对血管生成有不同的影响，这与它的链长有关。具体地说，通常为高分子量(HMW-HA)(例如，大于1×103 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个寡聚体)的HA则刺激血管生成。然而，关于衰老如何影响HA，以及这种影响是否会导致与衰老相关的愈合缺陷，人们知之甚少。更好地了解这一领域可能会导致改进的老年人伤口修复疗法的发展。在这一临床潜力的背景下，我们最近发现，从老年小鼠的结缔组织(肌腱)中提取的ECM提取物含有比年轻小鼠更多的HA。此外，我们注意到，老年小鼠的皮肤切除伤口(愈合速度比年轻小鼠的伤口要慢得多)和来自老年人类捐赠者的成纤维细胞培养产生的HA明显多于年轻小鼠。这些发现强调了进一步研究的必要性，目的是：(1)量化老化对伤口组织中HA浓度和分子量的影响，(2)确定细胞介导的过程(例如，HA合成/降解)对观察到的HA浓度和分子量的变化负责，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，这是已知在老年宿主中缺失的伤口修复的关键过程。这一建议将遵循以下两个假设：1)ECM中HA的年龄相关性增加伴随着HA分子谱的变化，这些变化不利于血管生成，但当受到刺激时，来自老年捐赠者的内皮细胞能够对促血管生成形式的HA产生反应。以下两个特定目标将解决这两个假设：(1)测定幼年、中年和老年小鼠真皮切除创面组织和培养的创面成纤维细胞中HA的浓度和分子量分布；(2)确定HA分子量类别(高、中、低)对体外血管生成的影响。在体外研究中，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萌发和从不同年龄的捐赠者分离的人微血管内皮细胞(HmECs)的管状生成。两者都是先前未经检验的HA和血管内皮细胞在衰老过程中的功能模型。这些目标和相关的特定目标将有助于我们的长期目标，即更好地了解ECM中与年龄相关的HA变化，以便检查在体内组织修复过程中利用内源性和外源性HA调节血管生成的效果。 与公共健康相关：我们认为透明质酸(HA)--D-葡萄糖醛酸/D-N-乙酰氨基葡萄糖二糖的简单聚合物--是细胞外基质的重要组成部分，在衰老的组织修复过程中调节血管生成。在这项提案中，我们将使用两个相关但独立的特定目标，使用动物和人类的衰老模型来验证以下假设：1)ECM中与年龄相关的HA增加伴随着HA分子谱的变化，这对血管生成不利，但当受到刺激时，2)来自老年供者的内皮细胞能够对促血管生成形式的HA做出反应。更好地了解细胞外基质中与衰老相关的HA的变化，将有助于研究在体内组织修复过程中利用内源性和外源性HA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.