Host neutrophils as direct mediators of tissue graft revascularization

 描述(申请人提供)：美国每年进行数百万次外科移植手术，以替换受损和/或病变的组织，包括皮肤、骨骼、神经和血管。不幸的是，不充分的血管重建仍然是一种常见的结果，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，有效地将移植物微血管系统与宿主循环系统连接起来。在此之前，我们发现人类血管细胞悬浮液在植入免疫缺陷小鼠体内后能有效地形成灌流的微血管(这里称为活性移植物)。相反，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血运重建效率低下的部分原因是成熟的血管系统天生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是由移植物血管系统分泌的因子及时控制的，这一过程在新生血管中上调，但随着血管系统的成熟而逐渐沉默。值得注意的是，我们发现，外源性提供由活跃移植物分泌的因子可以重新激活中性粒细胞活性，并增强原本无痛移植物的血运重建。此外，我们还确定了三个因素(IL-8， IL-6和CXCL1)可能推动这一过程，并发现阻断Notch信号可以重新激活这些细胞因子的表达，并增加中性粒细胞在惰性移植物中的募集。综上所述，我们的主要假设是，宿主中性粒细胞可以作为组织移植物血运重建的直接媒介。在这里，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，并将确定皮下移植到裸鼠体内对中性粒细胞募集和移植物血管形成的影响。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吻合口的形成。最后，我们将使用三种替代方法来确定阻断Notch信号是否能增强惰性移植物的血运重建：(I)抑制γ-分泌酶；(Ii)用诱骗Notch-1 Fc多肽抑制Notch；以及(Iii)诱导性基因消融Notch。在AIM-2中，我们将研究转化生长因子β在中性粒细胞激活中的作用。我们将通过将LysM-Cre小鼠与TGFBR2/loxP小鼠杂交，产生髓系缺乏转化生长因子β-R2的转基因小鼠；然后将这些转基因小鼠的中性粒细胞(转化生长因子β-R2-/-)转移到髓系耗竭的裸鼠中，以测试活跃移植物的血管形成能力。在……里面 此外，我们将使用我们的异种移植模型来评估提供体外转化生长因子β激活的中性粒细胞是否可以使人类组织移植物(即脂肪和骨移植物)血运重建。总而言之，这些研究将确定在移植物血管重建的背景下，特定的细胞因子和控制中性粒细胞招募和激活的因素。我们设想，利用中性粒细胞的血管重建潜力可以成为改进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.