Host neutrophils as direct mediators of tissue graft revascularization

 描述（由申请人提供）：每年，在美国进行数百万次外科移植手术，以替换受损和/或患病的组织，包括皮肤、骨骼、神经和血管。不幸的是，血运重建不足仍然是一个常见的结果，导致不同程度的移植物吸收和失败。我们的长期目标是开发一种策略，以有效地连接移植微血管系统的主机循环系统。先前，我们证明了人血管细胞的悬浮液在植入免疫缺陷小鼠后有效地形成灌注的微血管（本文称为活性移植物）。相比之下，成熟的血管网络无法与宿主血管系统连接（惰性移植物）。最近，我们发现惰性移植物的低效血运重建部分是由于成熟血管系统固有的不能与宿主中性粒细胞结合。 我们还发现，中性粒细胞的募集和激活及时控制的因子分泌的移植血管，这是一个过程，在新生血管上调，但随后逐渐沉默的血管成熟。值得注意的是，我们发现外源性提供活性移植物分泌的因子可以重新激活中性粒细胞活性，并增强惰性移植物的再血管化。此外，我们已经确定了三个因素（IL-8， IL-6和CXCL 1）可能驱动这一过程，并发现阻断Notch信号传导可以重新激活这些细胞因子的表达并增加惰性移植物中的中性粒细胞募集。综上所述，我们的总体假设是宿主中性粒细胞可以作为组织移植物血运重建的直接介质。在此，我们提出两个具体目标。在Aim-1中，我们将敲除活性移植物内皮中编码每个候选因子的基因，并将确定皮下植入裸鼠后对中性粒细胞募集和移植物血管化的影响。我们还将确定惰性移植物内皮中每个候选因子的过度表达如何影响宿主和供体血管之间的血管病变的形成。最后，我们将使用三种替代方法确定阻断Notch信号传导是否增强Indolent移植物的血管再生：（i）抑制γ-分泌酶;（ii）用诱饵Notch-1 Fc肽抑制Notch;和（iii）Notch的诱导型基因消融。在Aim-2中，我们将研究TGFβ在中性粒细胞活化中的作用。我们将通过将LysM-Cre小鼠与tgfbr 2/loxP小鼠杂交来产生骨髓谱系缺乏TGFβ-R2的转基因小鼠;然后我们将来自这些转基因小鼠（TGFβ-R2-/-）的中性粒细胞转移到骨髓耗竭的裸鼠中，以测试活性移植物获得血管化的能力。在 此外，我们将使用我们的异种移植物模型来评估提供离体TGFβ激活的中性粒细胞是否可以使人体组织移植物（即脂肪和骨移植物）再血管化。总的来说，这些研究将确定特定的细胞因子和因子管理移植物血运重建的背景下中性粒细胞的招募和激活。我们设想，利用中性粒细胞的血运重建潜力可能成为改善外科移植的新策略的基础。

 DESCRIPTION (provided by applicant): Every year, millions of surgical grafting procedures are performed in the United States to replace damaged and/ or diseased tissues, including skin, bones, nerves, and blood vessels. Unfortunately, inadequate revascularization remains a frequent outcome, leading to various degrees of graft resorption and failure. Our long-term goal is to develop a strategy to effectively connect a graft microvasculature to the host circulatory system. Previously, we showed that a suspension of human vascular cells efficiently forms perfused microvessels upon implantation into immunodeficient mice (referred to herein as an Active graft). In contrast, a mature vascular network fails to connect with the host vasculature (Indolent graft). More recently, we have found that the inefficient revascularization of Indolent grafts is in part due to the inherent inability of a mature vasculature to engage host neutrophils. We have also found that neutrophil recruitment and activation were timely controlled by factors secreted from the graft vasculature, a process that was upregulated in nascent vessels but was then progressively silenced as the vasculature matured. Of note, we showed that exogenous provision of factors secreted by an Active graft could reactivate neutrophil activity and enhance revascularization of an otherwise Indolent graft. Moreover, we have identified three factors (IL-8, IL-6, and CXCL1) potentially driving this process and found that blocking Notch signaling could reactivate the expression of these cytokines and increase neutrophil recruitment in Indolent grafts. Taken together, our overarching hypothesis is that host neutrophils can serve as direct mediators for the revascularization of tissue grafts. Here, we propose two specific aims. In Aim-1, we will knockout genes encoding each candidate factor in the endothelium of Active grafts and will determine the effect on neutrophil recruitment and graft vascularization upon subcutaneous implantation into nude mice. We will also determine how over-expressing each candidate factor in the endothelium of Indolent grafts affects the formation of anastomoses between host and donor vessels. Lastly, we will determine if blocking Notch signaling enhances the revascularization of Indolent grafts using three alternative approaches: (i) inhibition of γ-secretase; (ii) Notch inhibition with a decoy Notch-1 Fc peptide; and (iii) inducible genetic ablation of Notch. In Aim-2, we will study the role of TGFβ in neutrophil activation. We will generate transgenic mice in which the myeloid lineage lacks TGFβ-R2 by crossing LysM-Cre mice with tgfbr2/loxP mice; we will then transfer neutrophils from these transgenic mice (TGFβ-R2-/-) into myeloid-depleted nude mice to test the ability of Active grafts to get vascularized. In addition, we will use our xenograft model to evaluate if provision of ex vivo TGFβ- activated neutrophils can revascularize human tissue grafts (namely, fat and bone grafts). Collectively, these studies will determine specific cytokines and factors governing neutrophil recruitment and activation in the context of graft revascularization. We envision that harnessing the revascularization potential of neutrophils could become the basis for a new strategy to improve surgical grafting.