HGF Signaling in Adipose Tissue Growth and Metabolism

我们赞赏审查者对前一份提交材料所表现的极大热情。摘要指出，该申请涉及一个非常重要、有趣和研究不足的问题。该申请的显著优势是新颖，及时和重要的研究主题，高度的创新，坚实的初步数据，申请人的经验，以及根据先前的评审意见所做的改进数量。我们也感谢评审员对其他潜在问题和陷阱的持续深入的批评和洞察。我们已在当前提交的文件中解决了这些问题，该文件已密切关注两年期提案。 在对先前申请的批评中提出了三个要点。评审员对目标1中提出的体内研究非常热情，但担心C57和BALB/c裸鼠之间血管形成的遗传差异。评论者正确地指出，BALB/c小鼠具有较少的侧支血管，并且不像C57小鼠那样有效地重塑（例如参见：Chalothorn等人，Physiol Genomics 30：179-91; 2007），并且建议“HGF敲低和过表达的作用需要在相同的遗传背景下仔细解释”。我们完全同意，在解释我们的实验时，必须考虑血管发育的遗传差异。在目标#1中，我们将研究HGF敲低和HGF过表达对BALB/c裸鼠脂肪垫发育的影响，由于其免疫缺陷，这些裸鼠通常用于异种移植实验。尽管尚未在BALB/c裸鼠中研究侧枝发育，但我们预计其将受到限制，如在裸鼠来源的标准BALB/c小鼠中所观察到的那样。我们和其他人已经表明，血管化脂肪垫在BALB/c裸鼠中发育，因此不期望减弱的侧支循环。 在这些小鼠中的发育将阻止我们所提议的研究的完成。在这些实验中，小鼠遗传背景的影响对于HGF敲减和HGF过表达实验两者是相同的，这将简单地解释发现。 正如评审员所建议的，在目标#1中，我们还将在C57小鼠的皮肤下注射HGF敲低和过表达3 T3-F442 A的前脂肪细胞，并评估脂肪垫形成。我们先前已经在C57背景下在HGF过表达小鼠中进行了急性脂肪垫发育实验（Bell等人，Am J Physiol 294：E336-44;2008）。然而，目前尚不清楚植入的细胞在宿主体内停留超过72小时是否会在免疫活性的C57小鼠中诱导免疫应答，从而破坏发育中的脂肪垫。如果长期脂肪垫发育在C57小鼠中不可行，我们还可以在Nu/Nu小鼠（CrlLNU-Foxnlnu; Charles River）中进行实验，Nu/Nu小鼠是一种与任何原种或品系无关的远系繁殖免疫缺陷小鼠。Nu/Nu小鼠中的侧支发育可能更有效，正如在C57小鼠中观察到的那样。 第二个一般性批评是，目标2和3很重要，但不够完善。为了解决这一问题，我们将在目标#2中集中于测试具有和不具有活性HGF信号传导的前脂肪细胞作为周细胞稳定血管结构的能力的实验。拟议的实验 将使用前脂肪细胞单层系统作为唯一模型进行，以简化对结果的解释。这些实验虽然简化为两个细胞系统，但被认为是高度创新的。实验将系统地测试前脂肪细胞是否需要HGF来发挥周细胞的功能，以及成熟脂肪细胞是否不能作为周细胞来稳定血管结构。这些实验将填补我们对前脂肪细胞周细胞功能理解的一个重要空白， 这是进一步发展这一领域调查的重要基础。 第三个批评是“没有严格考虑系统的复杂性”。我们通过消除目标3，这是欠发达，并集中目标2直接测试HGF在前脂肪细胞周细胞功能的功能，解决了这一有效点。我们认识到，血管发育是一个复杂的过程，其中可能涉及许多因素。我们亦知道，在Aim中使用的两个组件系统， #2具有良好定义的优点，可能无法完全反映生长中脂肪组织中存在的细胞的复杂混合物。在解释目标2中的实验结果时，我们将牢记后一点，这将为脂肪组织生物学这一未充分研究的领域提供重要信息。 总体而言，已经取得的变化重点和加强这一建议，阐明通过HGF调节血管发育和前脂肪细胞周细胞功能，以促进脂肪组织生长的机制。拟议的研究可以在两年的资助期内完成，并将在一个新的研究领域提供重要的基础， 建议将以。

We appreciate the significant enthusiasm expressed by the reviewers for the previous submission. It was noted in the Summary that the application addresses a very important, interesting and understudied question. Noted strengths of the application were the novel, timely and significant research topic, the high degree of innovation, the solid preliminary data, the experience of the applicant, and the number of improvements made in response to prior reviewer comments. We also thank the reviewers for their continued thoughtful critique and insight into other potential problems and pitfalls. We have addressed these concerns in the current submission which has been tightly focused into a 2 year proposal. Three main points were raised in the critique of the previous application. The Reviewers were very enthusiastic about the in vivo studies proposed in Aim 1, but were concerned about genetic differences in vascularization between C57 and BALB/c nude mice. The reviewers correctly point out that BALB/c mice have less collateral blood vessels and do not remodel as efficiently as do C57 mice (for example see: Chalothorn et al, Physiol Genomics 30:179-91; 2007), and suggest that “effects of HGF knockdown and overexpression need to be interpreted carefully and on the same genetic background”. We fully agree that genetic differences in vascular development must be considered during interpretation of our experiments. In Aim #1 we will investigate the effect of both HGF knockdown and HGF overexpression on fat pad development in BALB/c nude mice, which are routinely used in xenotransplantation experiments due to their immunodeficiency. Although collateral development has not been studied in BALB/c nude mice, we expect that it will be limited as observed in standard BALB/c mice from which the nude mice were derived. We and others have shown that vascularized fat pads develop in BALB/c nude mice and thus do not expect that attenuated collateral development in these mice will prevent completion of our proposed studies. In these experiments, the effect of mouse genetic background will be identical for both the HGF knockdown and HGF overexpression experiments, which will simply interpretation of findings. As suggested by the reviewers, in Aim #1 we will also inject HGF knockdown and overexpressing 3T3-F442A preadipocytes under the skin of C57 mice and evaluate fat pad formation. We have previously carried out an acute fat pad development experiment in HGF overexpressing mice on the C57 background (Bell et al. Am J Physiol 294:E336-44;2008). However, it is not known if implanted cells left longer than 72 h in the host will induce an immune response in immunocompetent C57 mice that will destroy the developing fat pad. If long term fat pad development is not viable in C57 mice, we can also pursue experiments in Nu/Nu mice (CrlLNU-Foxn1nu; Charles River), an outbred immunodeficient mouse that is not associated with any stock or strain. Collateral development in Nu/Nu mice may be more efficient as has been observed for C57 mice. The second general criticism was that Aims 2 and 3 were significant but underdeveloped. To address this concern we will focus in Aim #2 on the experiments that test the ability of preadipocytes with and without competent HGF signaling to function as pericytes to stabilize vascular structures. The proposed experiments will be carried out using the preadipocyte monolayer system as the only model, in order to simplify interpretation of the findings. These experiments, although simplified to a two cell system, were considered highly innovative. Experiments will systematically test if HGF is required for preadipocytes to function as pericytes, and if mature adipocytes are unable to act as pericytes to stabilize vascular structures. These experiments will fill a significant gap in our understanding of preadipocyte pericytic function and provide an important base for further development of this area of investigation. The third criticism was that the “complexity of the system was not rigorously considered.” We address this valid point by elimination of Aim 3, which was underdeveloped, and focusing Aim 2 to directly test the function of HGF in preadipocyte pericytic function. We recognize that vascular development is a complex process in which many factors are likely involved. We also understand that the two component system to be used in Aim #2, which has the advantage of being well-defined, may not fully reflect the complex mixture of cells present in growing adipose tissue. We will keep this latter point in mind as we interpret the findings of experiments in Aim 2, which will provide important information in this understudied area of adipose tissue biology. Overall the changes that have been made focus and strengthen this proposal to elucidate the mechanisms through which HGF regulates vascular development and preadipocyte pericytic function to promote adipose tissue growth. The proposed studies can be completed within a two year funding period, and will provide an important foundation, in a novel area of research, on which future proposals will be based.