Signal Transduction in Models of Fibroblast Invasion

描述（由申请人提供）：伤口愈合是一个涉及多种细胞类型的复杂过程，但附近成纤维细胞侵入血栓被认为是限速步骤。反过来，成纤维细胞侵袭的速度在很大程度上受血小板和巨噬细胞释放的血小板衍生生长因子（PDGF）的作用控制。该建议的基础是探索数学建模的前景，可以用来弥合在受控实验条件下进行的分子/细胞水平细胞信号的体外研究与细胞群体行为动态进化的生理环境之间的基本差距。一般来说，如果靶向分子治疗策略是根据分子、细胞和组织水平的设计原则进行的，那么它们将具有最大的益处。基于先前和初步工作的指示，我们制定了一个新的假设模型，该模型具有重新定义成纤维细胞和类似细胞类型迁移的空间定向机制的潜力。我们的目标是通过实验和计算方法的结合，以定量的方式探索这一模型和替代模型。通过PI 3-激酶途径的细胞内信号传导将在活的成纤维细胞中进行监测，使用全内反射荧光（TIRF）显微镜，因为细胞感知并响应PDGF梯度。在这些实验中，形态极性和细胞迁移也将被跟踪，以定量地将迁移速度和转向行为与PI 3-激酶激活模式联系起来。初步研究表明，细胞极性在PI 3-激酶信号传导中施加了固有的偏倚，当细胞向上梯度迁移时，PDGF会加强这种偏倚。为了进一步测试这一机制，将使用各种分子扰动破坏细胞极性，并分析对PI 3-激酶信号传导的影响。最后，PI 3-激酶信号、细胞极性和细胞迁移之间的关系将被纳入一个有偏随机游走模型，该模型将最终定义成纤维细胞侵袭模型中的细胞通量项。这些努力将为更深入地研究控制生理过程的分子机制奠定基础。

DESCRIPTION (provided by applicant): Wound healing is a complex process involving multiple cell types, but invasion of nearby fibroblasts into the clot is considered the rate-limiting step. The rate of fibroblast invasion is in turn controlled, to a significant extent, by the action of platelet-derived growth factor (PDGF) released by platelets and macrophages in the clot. The basis of this proposal is to explore the prospect that mathematical modeling can be used to bridge the fundamental gap between in vitro studies of cell signaling at the molecular/cellular levels, performed under controlled experimental conditions, and physiological settings where cell population behaviors evolve dynamically. In general, targeted molecular therapeutic strategies will have the most benefit if they are informed by molecular-, cellular-, and tissue-level design principles. Based on indications from previous and preliminary work, we have formulated a new, hypothetical model with the potential for redefining the mechanisms by which migration of fibroblasts and similar cell types is spatially directed. We aim to explore this and alternative models in quantitative terms through a combination of experimental and computational approaches. Intracellular signaling through the PI 3-kinase pathway will be monitored in living fibroblasts, using total internal reflection fluorescence (TIRF) microscopy, as the cells sense and respond to PDGF gradients. In these experiments, morphological polarity and cell migration will also be tracked, in an effort to quantitatively relate migration speed and turning behavior to the pattern of PI 3-kinase activation. Preliminary work has shown that cell polarity imposes an intrinsic bias in PI 3-kinase signaling, which is reinforced by PDGF when cells migrate up-gradient. To further test this mechanism, cell polarity will be disrupted using a variety of molecular perturbations, and the effects on PI 3-kinase signaling will be analyzed. Finally, the relationships among PI 3-kinase signaling, cell polarity, and cell migration will be incorporated in a biased random walk model, which will ultimately define the cell flux term in a model of fibroblast invasion. These efforts will serve as a foundation for more in-depth studies of molecular mechanisms governing physiological processes.