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，透明质酸是二糖d -葡萄糖醛酸/ d - n -乙酰氨基葡萄糖胺的简单聚合物，它对血管生成的影响与其链长度有关。具体来说，天然HA，通常是高分子量的（HMW-HA）（例如，超过1 x 103 kDa）抑制血管生成，而低分子量的HA (LMW-HA)（例如，小于3 kDa/少于12低聚物）刺激血管生成。然而，对于衰老是如何影响血凝素的，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治疗方法的发展，伤口修复在老年人。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提取物比从年轻小鼠中获得的HA含量明显更高。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慢得多）和从老年人类供体培养的成纤维细胞产生的HA明显多于年轻小鼠。这些发现强调需要进一步的研究，以达到以下目标：(1)量化衰老对伤口组织中透明质酸浓度和分子量的影响，(2)确定导致任何观察到的透明质酸浓度和分子量变化的细胞介导过程（例如，透明质酸合成/降解），以及(3)确定透明质酸浓度和/或分子量的变化是否以及如何影响血管生成，这是一个对伤口修复至关重要的过程，已知老年宿主缺乏血管生成。该提案将遵循以下两个假设：1)内皮细胞间质膜中HA与年龄相关的增加伴随着HA分子量谱的变化，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促血管生成形式的HA做出反应。以下两个特定目标将解决这两个假设：（目标1）确定真皮切除伤口组织和从年轻、中年和老年小鼠分离的培养伤口成纤维细胞中的HA浓度和分子量谱；（目标2）确定HA分子量等级（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供体分离的人微血管内皮细胞（hmECs）的管生成。两者都是先前未经检验的HA和内皮细胞在衰老中的功能模型。这些目标和相关的特定目标将有助于我们的长期目标，即更好地理解细胞外基质中HA与年龄相关的变化，以检验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.