Molecular basis for defective pericyte-endothelial cell interactions regulating vascular malformations

到目前为止，毛细血管是最丰富的血管，与组织实质密切相连。 控制发育和病理疾病状态的细胞。它们由共同组装的内皮细胞组成 细胞(EC)与相关的周细胞形成管状网络。多年来，我们实验室一直在研究 EC管腔和管子组装的分子基础，以及EC衍生的分子 控制周细胞的募集、增殖和毛细血管基底膜的沉积，这一过程重新 停止EC-周细胞的相互作用。血管畸形，如动静脉畸形(AVM)和 海绵状畸形(CMS)(最常见于脑组织，称为CCM)，由一组 EC管形态发生显著异常并伴有壁细胞间相互作用缺陷的病理 特兹。一个关键点是，对潜在的分子基础缺乏根本的理解 从EC或周细胞的角度来看，这些畸形的发展。为了解决这些问题， 我们开发了两种新的血管畸形体外模型，一种使用人内皮细胞的类似AVM的模型 表达k-RAS激活突变(即k-RasV12)和利用表达k-RasV12的内皮细胞建立类CM模型 和T抗原(Tag)(以失调细胞周期)。在类AVM的情况下，内皮细胞明显加速管状 然而，与对照组内皮细胞相比，周细胞募集和基底膜沉积 与对照组相比大幅减少。在类CM的情况下，修饰的ECs形成大的囊泡(没有发芽- ING行为)，具有明显的EC增殖，而周细胞对EC表现出应答或不招募- 有衬里的包囊(在体内强烈模拟CMS)。因此，我们的两个体外模型都概括了在 VIVO的Avms和CMS以及其他初步数据进一步支持了这些结论。为了调查和 在体外与体内的研究结果相关联，我们正在利用缺失CCM1(selec-1)的CCM障碍的小鼠模型。 在有或没有EC共同诱导的情况下，以可诱导的方式在出生后的小鼠中激活突变- K-RAS或PI3蛋白的表达。初步数据表明，这种激活突变可以显著增强 CCM在体内的发育与EC中CCM1的缺失相结合，这支持我们的体外观察。 我们提出了三个具体的目标，以进一步研究血管性疾病的潜在分子基础。 并为这些疾病开发新的治疗方案；这些方案是： 目的#1：明确k-RasV12在ECs中的表达如何加速EC管的形成，但减少周长。 细胞-血管内皮细胞相互作用导致动静脉样畸形。 目的#2：明确k-RasV12在内皮细胞中的表达与CCM基因缺失和EC细胞周期调节的关系。 破裂导致海绵状畸形，周细胞募集明显不足。 目标#3：明确促炎介质如何影响周细胞-内皮细胞相互作用，以调节 依赖k-RasV12的动静脉样和海绵状畸形的稳定性。

Capillaries are by far the most abundant blood vessels and are critically interfaced with tissue parenchymal cells to control both development and pathologic disease states. They consist of co-assembled endothelial cells (EC) tube networks with associated pericytes. For many years, our laboratory has been investigating the molecular basis for EC lumen and tube assembly, as well as the mechanisms and EC-derived molecules that control pericyte recruitment, proliferation, and capillary basement membrane deposition, a process that re- quires EC-pericyte interactions. Vascular malformations, such as arteriovenous malformations (AVMs) and cavernous malformations (CMs) (most often observed in cerebral tissue, termed CCMs), constitute a group of pathologies with marked abnormalities in EC tube morphogenesis coupled to deficiencies in mural cell interac- tions. A critical point is that there is a fundamental lack of understanding of the underlying molecular basis for the development of these malformations from either the EC or pericyte perspective. To address these issues, we have developed two novel in vitro models of vascular malformations, an AVM-like model using human ECs expressing a k-Ras activating mutation (i.e. k-RasV12) and a CM-like model using ECs expressing k-RasV12 and T antigen (TAg) (to dysregulate the cell cycle). In the AVM-like case, the ECs markedly accelerate tube formation compared to control ECs, however, pericyte recruitment and basement membrane deposition is strongly reduced compared to controls. In the CM-like case, the modified ECs form large cysts (with no sprout- ing behavior) with evident EC proliferation, while pericytes show responsiveness or no recruitment to the EC- lined cysts (strongly mimicking CMs in vivo). Thus, both of our in vitro models recapitulate what is observed in vivo with AVMs and CMs, and other preliminary data further supports these conclusions. To investigate and correlate in vitro with in vivo findings, we are utilizing mouse models of CCM disorder that delete CCM1 (selec- tively within ECs) in an inducible manner in postnatal mice with or without EC co-induction of activating muta- tions in k-Ras or PI3 kinase. Preliminary data suggests that such activating mutations can markedly enhance CCM development in vivo in conjunction with EC deletion of CCM1, which support our in vitro observations. We propose three specific aims to further investigate the underlying molecular basis for vascular mal- formations and to develop new therapeutic options for these diseases; and they are: Aim #1: Define how k-RasV12 expression in ECs results in accelerated EC tube formation, but reduced peri- cyte-EC interactions leading to arteriovenous-like malformations. Aim #2: Define how k-RasV12 expression in ECs coupled with loss of CCM genes and EC cell cycle regula- tion leads to cavernous-like malformations with markedly deficient pericyte recruitment. Aim #3: Define how pro-inflammatory mediators affect pericyte-EC interactions to regulate the formation or stability of k-RasV12-dependent arteriovenous-like and cavernous-like malformations.