Collagen I, Hyaluronan, and Aging

描述（由申请人提供）：通常认为透明质酸（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-乙酰葡糖胺的简单聚合物-对血管生成具有与其链长相关的不同作用。具体地，天然HA，通常具有高分子量（HMW-HA）（例如，超过1 × 103 kDa）抑制血管生成，而低分子量HA（LMW-HA）（例如，小于3 kDa/小于12个寡聚体）刺激血管生成。然而，人们对衰老如何影响HA以及这种影响是否可能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改进的老年人伤口修复疗法的发展。在这种临床潜力的背景下，我们最近发现，ECM提取物的结缔组织（肌腱）的老年小鼠含有显着更大量的HA比从年轻的小鼠。此外，我们注意到，老年小鼠的皮肤切除伤口（其愈合速度比年轻小鼠的伤口慢得多）和从老年人供体培养的成纤维细胞产生的HA显著高于年轻小鼠。这些发现强调了对具有以下目标的进一步研究的需要：（1）定量老化对伤口组织中HA浓度和分子量的影响，（2）鉴定细胞介导的过程（例如，HA合成/降解），其负责任何观察到的HA浓度和分子量的变化，和（3）确定HA浓度和/或分子量的变化是否和如何影响血管生成，血管生成是已知在老年宿主中缺乏的伤口修复的关键过程。 该提案将遵循以下2个假设：1）ECM中HA的年龄相关性增加伴随着HA分子量谱的变化，这对血管生成有害，但当受到刺激时，2）来自老年供体的内皮细胞能够响应HA的促血管生成形式。以下2个特定目的将解决这2个假设：（目的1）确定从年轻、中年和老年小鼠中分离的皮肤切除伤口组织和培养的伤口成纤维细胞中的HA浓度和分子量特征，（目的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胶原中血管生成的互补模型：小鼠微血管外植体的发芽和从一系列年龄的供体中分离的人微血管内皮细胞（hmECs）的小管形成。两者都是以前未经检查的模型HA和内皮细胞功能的老化。 这些目标和相关的具体目标将有助于我们的长期目标，这是为了更好地了解ECM中HA的年龄相关变化，以检查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。 公共卫生相关性：我们提出透明质酸（HA）-一种简单的聚合物的二糖D-葡萄糖醛酸/D-N-乙酰葡糖胺-是细胞外基质的一个重要组成部分，调节血管生成在组织修复老化。在本提案中，我们将使用2个相关但独立的特定目标，采用动物和人类衰老模型来测试以下假设：1）ECM中HA的年龄相关增加伴随着HA分子量谱的变化，这对血管生成有害，但当受到刺激时，2）来自老年供体的内皮细胞能够对促血管生成形式的HA做出反应。更好地理解细胞外基质中HA的年龄相关变化将允许研究检查利用内源性和外源性HA在体内组织修复期间调节血管生成的作用。

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.