Beyond PECAM: Mechanisms of Transendothelial Migration

项目摘要 白细胞的跨内皮迁移（TEM）是炎症反应中的关键步骤，因为 大多数由于不希望的炎症引起的损伤发生在白细胞穿过血管之后。TEM是 受血小板/内皮细胞粘附分子-1（PECAM，CD 31）和CD 99等分子调节 在白细胞和内皮细胞（EC）边界上表达。在EC上，这些分子也存在于 一个相互连接的膜网状膜称为侧缘再循环室（LBRC） 其被“靶向”至TEM的位点。白细胞PECAM和EC PECAM之间的同种嗜性相互作用 控制渗出的开始。白细胞CD 99和EC CD 99之间的嗜同性相互作用是重要的 完成TEM。在本提案中，我们将利用上一个周期中获得的知识， 这些白细胞/内皮细胞相互作用招募LBRC以促进 有效的TEM和它们在白细胞上的分布是负责白细胞/EC 相互作用触发促进TR以促进TEM的信号通路。 在目的I中，我们将检验PECAM和CD 99在白细胞上的分布是 负责TEM的顺序调节。初步数据显示，在TEM过程中，PECAM是 CD 99主要集中在白细胞的前部，而CD 99主要集中在后部。我们怀疑这个浓度是至关重要的 PECAM和CD 99在内皮细胞上聚集。我们将在体外测试我们的假设， PECAM和CD 99的胞质结构域，以逆转它们在白细胞上的分布，并确定 这是否会影响它们调节TEM的顺序。我们还将测试它们是否调节TEM 使用旋转圆盘共聚焦活体显微镜连续地在体内研究该过程，以真实的时间。 CD 99 L2（L2）是我们最近发现的在TEM中起重要作用的分子。在Aim II中， 确定L2如何调节TEM。L2弥散分布于白细胞表面。我们将测试 假设它调节PECAM和CD 99调节之间的步骤，使用“顺序阻断” 我们已经开发并发布的检测方法。我们将识别L2用于执行以下操作的信号通路： 其功能并确定其是否在缺血/再灌注（I/R）损伤中起重要作用。我们将研究 通过活体显微镜观察小鼠提睾肌循环数小时内对I/R损伤的反应 心肌梗死模型。 我们最近确定了长期寻求的机制，内皮细胞CD 99信号在TEM。它 存在于激活蛋白激酶A（PKA）的多分子复合物中。在目标III中，我们将确定CD 99 PKA下游的信号通路。基于初步数据，我们假设Rac 1是一个关键的 PKA的下游效应子，以促进靶向再循环。我们将在两组受试者中检测CD 99在I/R损伤中的作用。 模型如上。

Project Summary Transendothelial migration (TEM) of leukocytes is a critical step in the inflammatory response, since most of the damage due to unwanted inflammation occurs after leukocytes cross blood vessels. TEM is regulated by molecules such as platelet/endothelial cell adhesion molecule-1 (PECAM, CD31) and CD99 expressed on both the leukocytes and at endothelial cell (EC) borders. On EC, these molecules also reside in an interconnected membrane reticulum of membrane called the lateral border recycling compartment (LBRC) that is “targeted” to the site of TEM. Homophilic interactions between leukocyte PECAM and EC PECAM control the start of diapedesis. Homophilic interactions between leukocyte CD99 and EC CD99 are important for the completion of TEM. In this proposal, we will build on the knowledge gained in the last cycle to test the overarching hypothesis that these leukocyte/endothelial cell interactions recruit the LBRC to promote efficient TEM and that their distribution on the leukocyte is responsible for the sequence in which leukocyte/EC interactions trigger signaling pathways that promote TR to facilitate TEM. In Aim I we will test the hypothesis that the distribution of PECAM and CD99 on the leukocyte is responsible for the sequential regulation of TEM. Preliminary data show that during TEM, PECAM is concentrated in the front of the leukocyte and CD99 at the rear. We suspect that this concentration is critical to clustering PECAM and CD99 on the endothelial cell. We will test our hypothesis in vitro by switching the cytoplasmic domains of PECAM and CD99 to reverse their distribution on the leukocyte and determine whether this affects the order in which they regulate TEM. We will also test whether they regulate TEM sequentially in vivo using spinning disc confocal intravital microscopy to study the process in real time. CD99L2 (L2) is a molecule we recently discovered to play an important role in TEM. In Aim II we will identify how L2 functions to regulate TEM. L2 is diffusely distributed on the leukocyte surface. We will test the hypothesis that it regulates a step between those regulated by PECAM and CD99 using the “sequential block” assay that we have developed and published. We will identify the signaling pathways used by L2 to carry out its function and determine whether it plays a significant role in ischemia/reperfusion (I/R) injury. We will study response to I/R injury by intravital microscopy in the cremaster muscle circulation over hours and in a mouse model of myocardial infarction over days. We recently identified the long-sought mechanism by which endothelial cell CD99 signals in TEM. It resides in a multimolecular complex that activates protein kinase A (PKA). In Aim III we will identify the CD99 signaling pathways downstream of PKA. Based on preliminary data we hypothesize that Rac1 is a critical downstream effector of PKA to promote targeted recycling. We will test the role of CD99 in I/R injury in the two models as above.