Anti-angiogenesis of HC-HA Covalent Complex and PTX3 Purified from Fetal Membrane

描述（由申请人提供）：血管生成是成人伤口愈合的一部分，从炎症开始，可能以瘢痕形成结束。当炎症、血管生成和瘢痕形成得到良好控制时，成人伤口愈合导致组织功能的恢复。相反，病理性血管生成，这是经常与不受控制的炎症可能会导致病理性瘢痕形成。在眼睛中，视觉是关键功能，任何这种容易形成疤痕的伤口愈合都可能导致失明。事实上，美国国立卫生研究院已经认识到，慢性伤口无法愈合是美国的一个主要健康问题，而且随着人口老龄化，这一问题的严重性将增加。长期以来，我们一直推测，解决上述问题的一种新方法在于更好地了解由羊膜（AM）和绒毛膜（CH）组成的胎膜的生物学功能。我们的推测部分来自AM移植在向眼表面递送抗炎、抗瘢痕形成和抗血管生成功效方面的累积临床成功，以及来自胎儿伤口愈合的奥秘，其缺乏炎症和血管生成，并且是“无瘢痕的”。我们已经成功地从AM中纯化了在透明质酸（HA）和间抑制剂（II）的重链（HC）之间形成的共价HC-HA复合物，并且已经将其鉴定为一种发挥抗炎、抗瘢痕形成和抗血管生成作用的活性组分。我们进一步收集了强有力的证据，支持从CH纯化的HC-HA比从AM纯化的HC-HA有效25倍，并且来自CH的PTX 3可以增强HC-HA对血管内皮增殖的抑制。因此，在该I期申请中，我们提出通过验证PTX 3确实是来自CH的增强HC“HA的抗血管生成作用的关键蛋白来描绘HC“HA的化学结构（目的1）;并表征和比较从CH纯化的HC“HA与从AM纯化的HC“HA的体外抗血管生成作用（目的2）。连同将由II期（R44 EY 017497）资助支持的研究，其集中于测试从AM纯化的HC“HA的体内抗炎和抗瘢痕形成功效，我们相信，成功完成上述两个目标将使我们首次提出新的治疗范例，其中单个HC“HA复合物可以同时缩短三个关键步骤，即，炎症、血管生成和成人伤口愈合的瘢痕形成。因此，我们设想HC“HA可能不仅是负责确保无瘢痕胎儿伤口愈合的胚胎基质，而且它还可以作为一种新的生物材料从胎膜中充分利用，我们可以从中推出新的治疗方法，使成人伤口愈合朝着再生方向发展。通过将PTX 3包括到HC-1HA中，我们可以开发出一类新的血管生成抑制剂，用于治疗眼科内外的许多疾病，其中病理性血管生成威胁我们的视力和健康。 公共卫生相关性：在该I期申请中描述的研究旨在开发一类新的血管生成抑制剂，其基于在透明质酸（HA）和间抑制剂的重链（HC）之间形成的共价HC“HA复合物，并从胎膜中纯化，即，羊膜和绒毛膜。我们提出的研究旨在证明PTX 3（一种已知发挥抗血管生成和抗炎作用的五聚蛋白家族成员）首先与从绒毛膜纯化的HC-HA强烈相关，其次负责增强HC-HA的抗血管生成效力。我们相信，PTX 3可以加入到HC-HA中以产生一类新的血管生成抑制剂，其不仅可以抑制bFGF和VEGF介导的血管生成，而且还可以通过减少额外的炎症和瘢痕形成而更有用和有效。因此，我们可以开发基于与HC-HA结合的PTX 3的新疗法，以治疗眼科内外的许多疾病，其中不需要的血管生成对我们的视力和健康有害。

DESCRIPTION (provided by applicant): Angiogenesis is a part of adult wound healing, which starts from inflammation and may end in scarring. When inflammation, angiogenesis, and scarring are well controlled, adult wound healing results in the restoration of tissue function. In contrast, pathologic angiogenesis that is frequently associated with uncontrolled inflammation may lead to pathologic scarring. In the eye, where vision is the key function, any such scar-prone wound healing can lead to blindness. In fact, the NIH has recognized that the inability of chronic wounds to heal is a major health problem in the United States and that such a problem will increase in magnitude as the population ages. We have long speculated that one novel solution to the aforementioned problem lies in a better understanding of the biological function of the fetal membrane, which consists of the amniotic membrane (AM) and the chorion (CH). Our speculation is derived partly from cumulative clinical successes of AM transplantation in delivering anti-inflammatory, anti- scarring, and anti-angiogenic efficacies to the ocular surface, as well as from the mystery of fetal wound healing that lacks inflammation and angiogenesis, and is "scarless". We have successfully purified the covalent HC"HA complex formed between hyaluronan (HA) and the heavy chain (HC) of inter- -inhibitor (I I) from the AM and has identified it as one active component that exerts anti-inflammatory, anti-scarring and anti-angiogenic actions. We have further gathered strong evidence supporting that HC"HA purified from the CH is 25 fold more potent than that purified from the AM and that PTX3 from the CH may enhance the HC"HA's suppression of vascular endothelial proliferation. In this Phase I application, we thus propose to delineate the chemical structure of HC"HA by verifying that PTX3 is indeed the key protein from CH to enhance HC"HA's anti-angiogenic action (Aim 1); and to characterize and compare the in vitro anti-angiogenic action of HC"HA purified from the CH to that from AM (Aim 2). Together with the study to be supported by a Phase II (R44 EY017497) grant, which focuses on testing in vivo anti-inflammatory and anti-scarring efficacies by HC"HA purified from the AM, we believe that successful completion of the above two aims will let us put forth a novel therapeutic paradigm, for the first time, where a single HC"HA complex can simultaneously curtail three key steps, i.e., inflammation, angiogenesis, and scarring of adult wound healing. As a result, we envision that HC"HA may not only be an embryonic matrix responsible for ensuring scarless fetal wound healing, but it can also be sufficiently harnessed from the fetal membrane as a novel biomaterial, from which we may launch new therapeutics to gear adult wound healing towards regeneration. By including PTX3 to HC"HA, we may develop a new class of angiogenesis inhibitors for treating many diseases in and beyond ophthalmology where pathologic angiogenesis threatens our vision and health. PUBLIC HEALTH RELEVANCE: The research described in this Phase I application is aimed at developing a novel class of angiogenesis inhibitors that are based on the covalent HC"HA complex formed between hyaluronan (HA) and the heavy chain (HC) of inter- -inhibitor and purified from the fetal membrane, i.e., the amniotic membrane and the chorion. Our proposed research intends to prove that PTX3, a pentraxin family member known to exert both anti-angiogenic and anti- inflammatory actions, first, is strongly associated with HC"HA purified from the chorion and, second, is responsible for enhancing the anti-angiogenic potency of HC"HA. We believe that PTX3 can be added to HC"HA to produce a new class of angiogenesis inhibitors, which will not only suppress angiogenesis mediated by bFGF and VEGF but will also be more useful and effective by curtailing additional inflammation and scarring. Consequently, we may develop new therapeutics based on PTX3 bound to HC"HA to treat many diseases in and beyond ophthalmology where unwanted angiogenesis is detrimental to our vision and health.