Vascular Pathogenesis of Port Wine Stain

项目总结 鲜红斑点(PWS)是一种先天性的、进行性的皮肤血管畸形，发生于 估计每1000名活产儿中有3-5名婴儿。在美国大约有120万人 各州都有PWS的胎记。脉冲染料激光(PDL)仍然是PWS的治疗选择。然而，只有 不到10%的患者在PDL后实现完全病变消退。PWS治疗结果不充分是一种 具有重要临床意义的问题，需要紧急解决。PWS的病因和发病机制是 目前对此知之甚少。这种知识差距是开发任何新的有效方法的主要障碍 治疗。PWS是NIAMS长期计划下的研究领域之一，被列为“了解 皮肤血管系统先天缺陷(如血管瘤和鲜红斑点)的原因和发展有效 治疗。“这里提出的研究是专门针对理解病理学的 这将有助于进一步研究PWS的机制，并促进开发新的、更有效的治疗方法。在……里面 在寻找PWS的致病物质时，我们已经发现循环中的CD31+外切体，它们是 来源于人皮损真皮微血管内皮细胞(HDMVECs)，促进 PWS的血管表型。对血清CD31+外切体蛋白质含量的分析显示，PWS CD31+外切体具有独特的分子环境，包括EphB1、EfnB2(EfnB2)、α-去整合素-和- 金属蛋白酶结构域30(ADAM30)和胞吐调节介质Synatotagamin like-1(SYTL1)。功能性 分析表明，ADAM30作为一种裂解EfnB2的脱水酶，其活性受血管调节 内皮生长因子受体2(VEGFR2)信号转导。更有趣的是，与正常对照组相比， PWS血清CD31+外切体和hDMVEC培养来源的EphB1/EfnB2/ADAM30外切体具有丰富的 更强的血管生成活性。他们大大增加了新血管的数量和大小 裸鼠体内异种移植模型。这些新发现促使我们检验一种假设：hDMVEC- 衍生的CD31+外切体在皮损部位丰富，并通过 通过分子途径破坏细胞-细胞和细胞-ECM相互作用和EC屏障功能 涉及外体EphB1/EfnB2/ADAM30(图1)。这些外切体也被释放到循环系统中。 它们可以被识别并作为潜在的血清生物标记物的系统。在目标1中，我们将确定 PWS ECs来源的CD31+外切体与正常对照相比的特异性分子图谱 体外和体内。我们将确定PWS hDMVECs的CD31+外切体的分泌是否受 胞吐基因SYTL1和Synatotagamin-1(SYT1)在体内和体外；在目标2中，我们将确定 EphB1/EfnB2调节VEGFR2信号激活ADAM30的机制 在目标3中，我们将确定PWS CD31+外体EphB1/EfnB2/ADAM30是否诱导 在体外形成PWS血管表型；在目标4中，我们将确定PWS CD31+外体 EphB1/EfnB2/ADAM30在体内诱导血管生成和PWS血管表型的形成。长的- 本研究的长期目标是探讨PWS的发病机制，以开发新的PWS治疗方法。 近期的目标是确定皮损内皮外切体在血管表型中的作用。 PWS的发病机制。我们建议的研究是完全新颖的，因此获得的数据将是重要的，因为它们 将为PWS的发病机制定义一条新的分子途径，并开辟新的途径 为PWS开发更有效的治疗策略。

PROJECT SUMMARY Port Wine Stain (PWS) is a congenital, progressive vascular malformation of human skin which occurs in an estimated incidence of 3-5 infants per 1,000 live births. Approximately 1.2 million individuals in the United States have PWS birthmarks. Pulsed dye laser (PDL) remains the choice of treatment for PWS. However, only less than 10% of patients achieve complete lesion fading after PDL. Inadequate PWS therapeutic outcome is a clinically significant problem that requires an urgent solution. The etiology and pathogenesis of PWS are currently poorly understood. This knowledge gap is a major obstacle for developing any new effective treatments. PWS is one of the research areas under the NIAMS long-range plan listed as “understanding the cause of skin vasculature birth defects (e.g., hemangioma and port wine stain) and developing effective therapies.” The studies proposed herein are specifically directed towards understanding pathological mechanisms of PWS and facilitating the development of new, more effective approaches to treatment. In searching for causative substances for PWS, we have identified that circulating CD31+ exosomes, which are derived from human lesional dermal microvascular endothelial cells (hDMVECs), facilitate the formation of vascular phenotypes of PWS. Analysis of the protein content of the serum CD31+ exosomes reveals that PWS CD31+ exosomes harbor a unique molecular milieu that include EphB1, ephrinB2 (EfnB2), a-disintegrin-and- metalloprotease domain 30 (ADAM30), and exocytosis mediator synatotagamin like-1 (SYTL1). Functional analysis shows ADAM30 functions as a sheddase to cleave EfnB2 and its activity is regulated by vascular endothelial growth factor receptor 2 (VEGFR2) signaling. More interestingly, compared to normal controls, PWS serum CD31+ exosomes and hDMVEC culture-derived EphB1/EfnB2/ADAM30 exosomes possess much greater angiogenesis activity. They greatly increased the number and size of new blood vessels formed in a nude mouse xenograft model in vivo. These novel findings motivate us to test a hypothesis that hDMVEC- derived CD31+ exosomes enrich at lesional sites and cause the development and progression of PWS via disruption of cell-cell and cell-ECM interactions and EC barrier function through a molecular pathway involving exosomal EphB1/EfnB2/ADAM30 (Fig. 1). These exosomes are also released into the circulatory system where they can be identified and serve as potential serum biomarkers. In Aim 1, we will determine the specific molecular profiles of CD31+ exosomes derived from PWS ECs as compared to normal controls in vitro and in vivo. We will determine if secretion of CD31+ exosomes from PWS hDMVECs is regulated by exocytosis genes SYTL1 and synatotagamin-1 (SYT1) in vivo and in vitro; In Aim 2, we will determine the mechanisms by which EphB1/EfnB2 regulates VEGFR2 signaling to activate ADAM30 leading to subsequent EfnB2 cleavage; in Aim 3, we will determine if PWS CD31+ exosomal EphB1/EfnB2/ADAM30 induces the formation of PWS vascular phenotypes in vitro; and in Aim 4, we will determine if PWS CD31+ exosomal EphB1/EfnB2/ADAM30 induces angiogenesis and formation of PWS vascular phenotypes in vivo. The long- term goal of this research is to investigate the pathogenesis of PWS for developing new treatments of PWS. The immediate goal is to establish the role of lesional endothelial exosomes in the vascular phenotypes in the pathogenesis of PWS. Our proposed studies are totally novel and data so obtained will be important since they will define a novel molecular pathway for the pathogenesis of PWS and open new avenues for the development of more effective therapeutic strategies for PWS.