Engineering Growth Factor-ECM Interactions to Heal Chronic Wounds in Type 1 Diabetes

 描述：我们寻求开发和评估新的生物材料/生长因子疗法来治疗糖尿病足溃疡，糖尿病足溃疡是糖尿病患者的主要并发症，导致大量并发症，包括截肢、寿命和生活质量下降。在目前的临床实践中，生长因子疗法普遍令人失望：基于我们已发表和未发表的初步工作，我们认为生长因子疗法总体上的主要问题是生长因子-细胞外基质(ECM)微环境的进化背景被忽视。我们的团队已经阐明了生长因子和细胞外基质蛋白质之间复杂的生物分子相互作用，这将极大地改变生长因子的生物学特性。事实上，我们已经开始相信，大多数生长因子进化为与ECM蛋白协同发挥作用(Martino等人，Science Transl.地中海医院。2011年，以及其他参考文献)。在这里，我们将评估生长因子工程和ECM工程方法，这些方法旨在控制糖尿病创面愈合中生长因子和ECM之间的动态相互作用。一方面，我们将设计与细胞外基质具有超亲和力的生长因子变异体，包括我们为细胞外基质工程选择的翻译生物材料平台纤维蛋白；我们将设计细胞外基质，使其与生长因子具有高亲和力和混杂。我们将在小鼠1型糖尿病(T1D)的延迟愈合模型中探索与糖尿病足部溃疡相关的反应，建立一个小鼠诊所，在该诊所中，动物接受胰岛素治疗，以控制高血糖但不能恢复正常的正常血糖，以模拟患者不完全的血糖控制。(特定目标1)我们将使用蛋白质工程的方法在NOD小鼠伤口愈合延迟的模型中，以血管内皮生长因子-A诱导血管生成的能力，PDGF-BB和HB-EGF诱导再上皮化的能力，以及CXCL-12的诱导能力为靶点，利用ECM超亲和变异体血管内皮生长因子-A、PDGF-BB、HB-EGF和CXCL-12 祖细胞浸润。具体地说，根据我们的初步工作(Martino等人，科学2014)，一个来自PlGF-2的高亲和力和混杂的ECM结合结构域(PlGF-2123-144)将与这些因子融合，产生VEGF-A-PlGF-2123-144、PDGF-BB-PlGF-2123-144、HB-EGF-PlGF-2123-144和CXCL12-PlGF-2123-144。我们提供了在糖尿病小鼠T2D创面模型中联合使用VEGF-A-PlGF-2123-144，PDGF-BB-PlGF-2123-144，双因素治疗的初步数据。(具体目标2)我们将在纤维蛋白中使用一种新型的蛋白酶抑制剂来稳定这一点，这是一种人类蛋白，用来取代目前用于纤维蛋白密封剂中的牛抑肽酶，以避免重复使用时的免疫并发症。(具体目标3)最后，我们将利用我们在von Willebrand因子(vWF1328-1351)中发现(未发表)的与纤维蛋白结合的高亲和力混杂生长因子结合域来创建与天然和工程生长因子的高亲和力结合位点。我们的目标是生产一种稳定的纤维蛋白基质，含有一种或两种生物活性因子，用于治疗T1D患者的伤口。

 DESCRIPTION: We seek to develop and evaluate novel biomaterial/growth factor therapies to heal diabetic foot ulcers, which plague diabetes patients as a major diabetes complication, resulting in substantial morbidity including amputation and reduction of lifespan and quality of life. In current clinical practice, growth factor therapies have generally been a disappointment: Based on our published and unpublished preliminary work, we believe that the major problem with growth factor therapeutics in general is that the evolutionary context of the growth factor-extracellular matrix (ECM) microenvironment has been ignored. Our group has elucidated complex biomolecular interactions between growth factors and proteins of the ECM, which dramatically alter the biology of the growth factor. Indeed, we have come to believe that most growth factors evolved to function in concert with an ECM protein (Martino et al., Science Transl. Med. 2011, among other references). Here, we will evaluate both growth factor engineering and ECM engineering approaches directed to control the dynamic reciprocity between growth factors and the ECM in diabetic wound healing. We will on the one hand engineer growth factor variants to bind with super-affinity to the ECM, including fibrin, our selected translational biomaterial platform for ECM engineering; and we will engineer the ECM to bind with high affinity and promiscuity to growth factors. We will work in a mouse type 1 diabetes (T1D) model of delayed healing to explore responses relevant to diabetic foot ulcers, establishing a mouse clinic in which animals are treated with insulin so as to control hyperglycemia yet not restore normal euglycemia, to model imperfect glycemic control in patients. (Specific Aim 1) We will employ a protein engineering approach to engineer ECM super-affinity variants of VEGF-A, PDGF-BB, HB-EGF and CXCL12 in the NOD mouse model of delayed wound healing, targeting VEGF-A for its ability to induce angiogenesis, PDGF-BB and HB-EGF for their ability to induce re- epithelialization, and CXCL-12 for its ability to induce progenitor cell infiltration. Specifically, based on our preliminary work (Martino et al., Science 2014), a high affinity and promiscuous ECM binding domain from PlGF-2 (PlGF-2123-144) will be fused to these factors, to yield VEGF-A-PlGF-2123-144, PDGF-BB-PlGF-2123-144, HB-EGF-PlGF-2123-144 and CXCL12-PlGF-2123-144. We present preliminary data with combined VEGF-A-PlGF- 2123-144, PDGF-BB-PlGF-2123-144, dual factor therapy in a murine T2D model of diabetic wounds. (Specific Aim 2) We will do this in fibrin stabilized with a novel protease inhibitor, a human protein to replace the bovine aprotinin currently used in fibrin sealants, to avoid immunological complication upon repeated use. (Specific Aim 3) Finally, we will utilize a high affinity promiscuous growth factor binding domain that we have discovered (unpublished) in von Willebrand factor (vWF1328-1351) bound in fibrin to create high affinity binding sites to both native and engineered growth factors. Our objective is to produce a stabilized fibrin matrix, with one or perhaps two bioactive factors, to heal wounds in T1D patients.