Paracrine IGF/GFBP Interactions In Vivo

描述（由申请人提供）：胰岛素样生长因子（IGFs）调节细胞增殖、存活和分化。这些作用是由当地产生的igf以及来自环流的igf进行的。IGF在血浆和细胞间室之间的分配可能部分是由高亲和力IGF结合蛋白家族成员完成的。我们认为，在体内组织特异性扰动后，旁分泌IGF-I和igfbp的功能得到了最好的探索，因为它们的活性似乎在损伤或其他触发组织重塑后被激活。当受到特殊病理生理挑战时，血管壁、肠和膀胱的SMC保留了增殖和重塑细胞外基质的能力。igf - 1是其中最突出的，因为它能刺激SMC的生长和体外和体内存活。球囊动脉内损伤后，igf - 1基因表达明显诱导。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，促进了新内膜的形成。尽管如此，旁分泌分泌IGF-I作为损伤反应介质的必要性尚未最终确定，其他因素决定局部IGF-I生物利用度的作用也尚未确定。在本提案中，我们将使用小鼠遗传策略来解决其中一些问题。我们将追求以下目标：1)确定平滑肌对损伤的反应是否需要Igflr：我们将首先表征平滑肌特异性缺失IGF I型受体（Igflr）对动脉损伤后生长、迁移和新内膜形成的影响。2)确定血管损伤对旁分泌生成lGF-1的需求：我们将在SMP8-cre-ERT2小鼠与loxP-Igfl小鼠纯合子杂交中探讨这个问题。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)确定平滑肌损伤反应中IGFBP局部表达的需求：我们将首先通过依赖他莫昔芬的方式在平滑肌细胞中靶向缺失lGFBP-4来探索这一点。我们将验证SMP8-cre-ERT2/loxP-lgfbp-4小鼠会减少平滑肌组织中IGF-I的储存，这将与颈动脉损伤的反应受损有关。4)确定锌金属蛋白酶PAPP-A在平滑肌损伤反应中局部表达的需求：我们将验证他莫昔芬诱导的重组后，IGFBP蛋白酶活性被消除或抑制，IGFBP-4稳定，导致IGF-I被隔离，SMC迁移和增殖受损的假设。

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.