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 ?-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 TGF? in neutrophil activation. We will generate transgenic mice in which the myeloid lineage lacks TGF?-R2 by crossing LysM-Cre mice with tgfbr2/loxP mice; we will then transfer neutrophils from these transgenic mice (TGF?-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 TGF?- 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.
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. Here, we propose studies to better understand the mechanisms by which host neutrophils facilitate the revascularization of human grafts. We envision this research will pave the way for a new strategy to improve surgical grafting.
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