Guidance cues and pattern prediction in the developing retinal vasculature: A combined experimental and theoretical modelling approach

该项目的目的是使用最新的数学建模（MM）技术和最先进的3d成像技术来发现小鼠视网膜脉管丛（RVP）的最终模式是如何在正常发育的小鼠和具有严重血管缺陷的小鼠（vegf -转基因）中进行调节的；类似于那些导致人类婴儿终生失明的物质。随着视网膜的生长，它的代谢需要得到三维血管网络的支持，这些血管网络形成了一种典型的毛细血管网络模式，连接着视网膜不同层的小动脉和静脉。RVP的最终结构是由组织中的分子途径决定的，内皮细胞（形成血管的主要细胞）和周细胞（赋予血管结构/功能稳定性的细胞）在这些分子途径上迁移。这些细胞的迁移方向取决于可溶性因子和基质相关因子的浓度梯度，这些因子在化学上（趋化性）将细胞吸引到视杯边缘。使用共聚焦和双光子显微镜（允许收集和同化细胞、通路和趋化剂的三维图像），我们将检查正常和新生血管畸形小鼠不同发育阶段的视网膜，以发现哪些分子通路和趋化剂在决定RVP的模式和最终成熟中至关重要。这些图像是通过用特定的染料标记细胞（例如内皮细胞用一种叫做BSI-B4的隔离素；周细胞用一种针对平滑肌肌动蛋白的探针和带有特定抗体的通路/趋化剂），用激光刺激组织，并用共聚焦显微镜捕获荧光染料发出的光而产生的。由于在这些研究中收集的所有图像基本上都是RVP生长过程中某个特定时间发生的事情的快照，因此有必要能够覆盖这些结果并发现细胞如何响应随着时间（暂时）产生的通路和趋化剂的潜在表达模式。这就是MM的强大工具可以导致关于如何在组织，细胞和分子水平上调节事件组合的新发现的地方。第一个拟议的MM最初将依赖于在正常和vegf转基因新生小鼠中进行的研究数据。在收集、数字化和量化一系列参数（即血管长度、分支点、分形维数（在不同尺度上重复基本模式的频率）、细胞类型、位置和分子浓度）之后，这些信息被用于通知MM，以便生成RVP的虚拟模型。然后通过测试其在开发后期阶段预测RVP模式的能力来验证和改进该MM。MM可以预测生物学数据（通路和趋化梯度）中的空白，然后通过提出新的研究来为生物学实验提供信息，这些研究将进一步阐明RVP三维结构发展背后的细胞和分子机制。这是生物学家和数学家之间的一次独特合作，在发现复杂的3d组织RVP是如何生长并对正常动物和患有严重眼部病变的动物的最终眼睛结构做出贡献方面，这两个学科都具有指导意义。该项目的最后一部分将使用MM来预测治疗vegf转基因小鼠的治疗方法，以防止眼部病理的进展。这项研究将有助于基本的生物学理解血管如何在3d中生长（这决定了所有器官的生长），眼睛如何正常生长，更具体地说，在以血管形成不适当为特征的眼部状况下（所有导致新生儿和成人失明的主要疾病）。

Summary The aim of this project is to use the latest mathematical modelling (MM) techniques coupled with state-of-the-art 3-D imaging to discover how the final patterning of the mouse retinal vasculature plexus (RVP) is regulated in both normally developing mice and mice with severe vascular defects (VEGF-transgenics); similar to those observed to cause a lifetime of blindness in human babies . As the retina grows, its metabolic needs are supported by a 3-D network of blood vessels that form a characteristic pattern of capillary networks linking the arterioles and veins in its different layers. The final structure of the RVP is determined by molecular pathways in the tissue, on which the endothelial cells (the main cell forming the blood vessels) and pericytes (cells which give structural/functional stability to the vessels) migrate. The direction of migration of these cells is dependent on the concentration gradients of both soluble and matrix-associated factors which chemically (chemotaxis) attract the cells towards the rim of the optic cup. Using confocal and 2-photon microscopy (allowing the collection and assimilation of 3-D images of cells, pathways and chemotactic agents) we will examine the retina from different developmental stages of normal and neonatal mice with vascular malformations to discover which molecular pathways and chemotactic agents are critical in determining the patterning and final maturation of the RVP. The images are generated by labelling cells with specific dyes (for instance endothelium with an isolectin called BSI-B4; perictes, with a probe against smooth muscle actin and pathways/chemotactic agents with specific antibodies) exciting the tissue with a laser and capturing the light from the fluorescing dyes with a confocal microscope. As all the images collected in these studies are essentially snapshots of what happens at one particular time during the growth of the RVP, it is essential to be able to overlay these results and discover how the cells respond to the underlying expression patterns of the pathways and chemotactic agents being produced over time (temporally). This is where the powerful tool of MM can lead to new discoveries about how the combination of events (at the tissue, cell and molecular level) are regulated. The first proposed MM will initially rely on data generated from studies performed in normal and VEGF-transgenic neonatal mice. After collecting, digitising and quantitating a series of parameters (i.e. vessel lengths, branch-points, fractal dimension [how often a basic pattern is repeated at different scales], cell-type, location and concentration of molecules) this information is used to inform the MM, so that a virtual model of the RVP can be generated. This MM is then verified and improved, by testing its ability to predict the RVP patterning at later stages during development. Gaps in the biological data (in both the pathways and the chemotactic gradients) can be anticipated by the MM that will then be used to inform biological experiments by proposing new studies which will further elucidate the cellular and molecular mechanisms underlying the development of the 3-D structure of the RVP. This is a unique collaboration between biologists and mathematicians, in which both disciplines are instructive in discovering how a complex 3-D tissue, the RVP, grows and contributes to the final structure of the eye in both normal animals and animals with a serious ocular pathology. The final portion of the project will employ the MM to predict which therapeutic approaches to treat the VEGF-transgenic mice, will prevent progression of ocular pathology. This research will benefit basic biological understanding of how blood vessels grow in 3-D (which determines the growth of all organs), how the eye grows normally and more specifically in ocular conditions characterised by inapprpriate blood vessel formation (all major diseases that cause blindness in neonates and adults).