Paracrine IGF/GFBP Interactions In Vivo

描述（由申请人提供）：胰岛素样生长因子（IGF）调节细胞增殖、存活和分化。这些行动是由当地生产的IGFs以及来自流通的IGFs进行的。IGF在血浆和细胞间质区室之间的分配可能部分由高亲和力IGF结合蛋白家族的成员完成。我们认为旁分泌IGF-I和IGFBPs的功能最好在体内组织特异性扰动后进行研究，因为它们的活性似乎在损伤或其他组织重塑触发因素后被招募。血管壁、肠和膀胱的SMC在经受特定病理生理挑战时保留增殖和重塑细胞外基质的能力。IGF-I在其中是突出的，因为它刺激SMC在体外和体内的生长和存活。球囊损伤后，IGF-I基因的表达有明显的诱导作用。此外，在SMC中靶向过表达IGF-I的转基因小鼠通过增加颈动脉损伤后的增殖和细胞迁移来增强新生内膜形成。尽管如此，IGF-1作为损伤反应介质的旁分泌产生的需要尚未最终确定，也没有确定其他因素决定局部IGF-1生物利用度的作用。在本提案中，我们将使用小鼠遗传策略来解决其中的一些问题。我们将追求以下目标：1）确定lgflr是否是平滑肌对损伤的反应所需的：我们将首先表征IGF I型受体（Igflr）的平滑肌特异性缺失对动脉损伤后生长、迁移和新生内膜形成的影响。2)确定响应于血管损伤的IGF-1的旁分泌产生的需求：我们将在与纯合loxP-Igfl小鼠杂交的SMP 8-cre-ERT 2小鼠中探索这个问题。损伤实验将在他莫昔芬诱导IgfI等位基因缺失和局部IGF-I组织储存耗尽后进行。3)确定平滑肌对损伤的反应中局部IGFBP表达的需求：我们将首先通过以他莫昔芬依赖性方式靶向缺失平滑肌细胞中的IGFBP-4来探索这一点。我们将检验SMP 8-cre-ERT 2/loxP-lgfbp-4小鼠将具有减少的IGF-I的平滑肌组织储存，并且这将与对颈动脉损伤的受损反应相关的假设。4)确定在平滑肌对损伤的反应中锌金属蛋白酶PAPP-A的局部表达的需要：在这里，我们将检验以下假设：在他莫昔芬诱导的floxed PAPP-A重组后，IGFBP蛋白酶活性将被消除或抑制，IGFBP-4稳定，导致IGF-I隔离和SMC迁移和增殖受损。

DESCRIPTION (provided by applicant): Insulin-like growth factors (IGFs) regulate cell proliferation, survival and differentiation. These actions are carried out by locally produced IGFs as well as IGFs derived from the circulation. IGF partition between plasma and cellular interstitial compartments is likely accomplished in part by members of a family of high affinity IGF binding proteins. We believe the function of paracrine IGF-I and of the IGFBPs is best explored after tissue-specific perturbations in vivo, since their activity appears to be recruited after injury or other triggers for tissue remodeling. SMC of the vascular wall, intestine and urinary bladder retain the ability to proliferate and to remodel the extracellular matrix when subjected to particular pathophysiological challenges. IGF-I is prominent among them, as it stimulates SMC growth and survival in vitro and in vivo. After balloon endoarterial injury, there is a marked induction of IGF-I gene expression. Moreover, transgenic mice with targeted overexpression of IGF-I in SMC have enhanced neointimal formation through increased proliferation and cell migration after carotid artery injury. Despite this, the requirement for paracrine production of IGF-I as a mediator of injury responses has not been conclusively established, nor is the role of other factors determining local IGF-I bioavailability. In this proposal we will address some of these questions using mouse genetic strategies. We will pursue the following aims: 1) Determine whether lgflr is required for the smooth muscle response to injury: We will first characterize the effects of smooth muscle-specific deletion of the IGF type I receptor (Igflr) on growth, migration and neointimal formation after arterial injury. 2) Determine the requirement of paracrine production of lGF-1 in response to vascular injury: We will explore this question in SMP8-cre-ERT2 mice crossed with homozygous loxP-Igfl mice. Injury experiments will be done after tamoxifen-induced deletion of the IgfI alleles and depletion of local IGF-I tissue stores. 3) Determine the requirement for local IGFBP expression in smooth muscle response to injury: We will first explore this by targeted deletion of lGFBP-4 in smooth muscle cells in a tamoxifen-dependent manner. We will test the hypothesis that SMP8-cre-ERT2/loxP-lgfbp-4 mice will have diminished smooth muscle tissue stores of IGF-I, and that this will be associated with impaired responses to carotid artery injury. 4) Determine the requirement for local expression of the zinc metalloproteinase PAPP-A in smooth muscle responses to injury: Here we will test the hypothesis that after tamoxifen-induced recombination of floxed PAPP-A, IGFBP protease activity will be abolished or dampened, IGFBP-4 stabilized, resulting in IGF-I sequestration and impaired SMC migration and proliferation.