Capillary malformation: From somatic GNAQ mutations to disrupted endothelial biology

毛细血管畸形：从体细胞 GNAQ 突变到内皮生物学破坏

• 批准号: 10058384
• 负责人: Joyce E. Bischoff
• 金额: $ 88.94万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058384
• 关键词: ANGPT2 gene; Address; Affect; Alleles; Angiopoietin-2; Animal Model; Automobile Driving; Bar Codes; Biology; Blood Vessels; Blood capillaries; Brain; Cell model; Cell physiology; Cells; Choroid; Cognitive; Collaborations; Coupled; Cutaneous; Data; Defect; Dermis; Development; Endothelial Cells; Endothelium; Event; Experimental Models; Eye; Face; Functional disorder; G alpha q Protein; G-substrate; GNAQ gene; Genetic Transcription; Genomics; Genotype; Glaucoma; Goals; Growth; Hematopoietic; Hemorrhage; Heterogeneity; Heterotrimeric G Protein Subunit; Histologic; Human; Image; Impairment; Implant; In Situ Hybridization; In Vitro; Infant; Inflammatory; Knock-in; Lead; Leptomeninges; Lesion; Malignant Neoplasms; Massachusetts; Mediator of activation protein; Medical; Medical center; Modeling; Molecular; Morphogenesis; Mus; Mutation; NF-kappa B; Neurocutaneous Syndromes; Neurologic; Nodule; Nude Mice; Pathogenesis; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Phospholipase; Play; Port-Wine Stain; Protein Kinase C; Reporting; Resistance; Retinal Detachment; Role; Seizures; Signal Transduction; Skin; Small Interfering RNA; Somatic Mutation; Specimen; Stains; Sturge-Weber Syndrome; Surface; TNFSF5 gene; Tamoxifen; Technology; Testing; Time; Tissue Engineering; Transcript; Universities; Work; Zebrafish; causal variant; cell type; cerebral capillary; drug candidate; drug testing; high risk; improved; in vivo; insight; knock-down; malformation; mouse model; mutant; nervous system disorder; novel; prevent; response; shear stress; single-cell RNA sequencing; skeletal; small hairpin RNA; small molecule inhibitor; soft tissue; stroke-like episode; tissue repair; transcriptome sequencing; venule

Project Abstract Our studies are focused on capillary malformation (CM) (previously referred to as “port-wine stain”), the most common type of vascular malformation. CM, excessive, enlarged capillary-like vessels just below the surface of the skin, are sporadic congenital lesions that darken, form nodules, and cause soft-tissue and skeletal overgrowth beneath the stain. Sturge-Weber syndrome (SWS) is a neurocutaneous disorder associated with CMs of the face, leptomeninges, and the choroid of the eye; patients suffer from neurological defects and glaucoma. Importantly, drug treatment for CMs does not exist and there is no cure. The 2013 discovery of a somatic activating mutation in GNAQ (p.R183Q) in non-syndromic cutaneous CMs and SWS CMs set the stage for molecular studies of this understudied vascular malformation. GNAQ encodes Gαq, the α-subunit of the heterotrimeric Gq protein that activates phospholipase Cβ. We showed that the GNAQ R183Q allele is enriched in the endothelial cell (EC) sorted from cutaneous CM and SWS brain specimens. We have worked on creating cellular and mouse models to elucidate how the GNAQ mutation affects EC function, how these alterations lead to CM, and how we can prevent the formation or growth of CM. We show that human ECs with the R183Q mutation do not respond properly to laminar shear stress, fail to form an endothelial barrier, and form enlarged CM-like vessels when implanted into mice. We implicate protein kinase C (PKC) and angiopoietin-2 (ANGPT2) as potential targets to reverse the GNAQ R183Q-driven CM. We are making strong progress towards an inducible, endothelial-specific knock-in of Gnaq R183Q in mice in which we have found CM-like lesions upon tamoxifen-induced expression of the knocked-in mutant allele. In this proposal we will identify the breadth of cell types that carry the somatic GNAQ R183Q allele and how the mutation alters the transcriptional profile versus non-mutant cells of the same phenotype (Aim 1). We will develop novel animal models in mice and zebrafish to elucidate the cellular steps leading to CM and will use them as platforms for testing candidate drugs (Aim 2). We will deeply interrogate the role of (ANGPT2) as a downstream functional mediator of constitutively active, mutant Gαq (Aim 3). These studies will deepen our understanding of how Gαq activity participates in capillary morphogenesis, result in the first animal models for CM/SWS, and provide a platform to test drugs that can prevent or regress CM. Discoveries about the pathophysiology of CM will also help us understand the mechanisms that underlie additional vascular lesions and improve our ability to identify new pathways for preventing vascular overgrowth (e.g., cancer) and promoting vascular growth during tissue repair or engineering.

项目摘要 我们的研究主要集中在毛细血管畸形（CM）（以前称为“葡萄酒斑”）， 血管畸形的常见类型。CM，表面下过度、扩大的毛细血管样血管 是散发性的先天性病变，会变暗，形成结节，并导致软组织和骨骼 污渍下的过度生长Sturge-Weber综合征（SWS）是一种神经皮肤疾病， 面部、软脑膜和眼脉络膜的CM;患者患有神经缺陷， 青光眼重要的是，CM的药物治疗不存在，也没有治愈方法。 2013年在非综合征性皮肤CM中发现GNAQ（p.R183Q）的体细胞激活突变 SWS CM为这种未充分研究的血管畸形的分子研究奠定了基础。GNAQ编码 Gαq，异源三聚体Gq蛋白的α亚基，可激活磷脂酶Cβ。我们证明了 GNAQ R183 Q等位基因在从皮肤CM和SWS脑中分选的内皮细胞（EC）中富集 标本我们一直致力于创建细胞和小鼠模型，以阐明GNAQ突变是如何 影响EC功能，这些改变如何导致CM，以及我们如何预防CM的形成或生长。 我们发现，具有R183 Q突变的人内皮细胞对层流剪切应力没有适当的反应， 形成内皮屏障，并在植入小鼠体内时形成扩大的CM样血管。我们认为蛋白质 激酶C（PKC）和血管生成素-2（ANGPT 2）作为逆转GNAQ R183 Q驱动的CM的潜在靶点。 我们在小鼠中的Gnaq R183 Q的诱导性、内皮特异性敲入方面取得了很大进展， 我们已经发现在他莫昔芬诱导的敲入突变等位基因表达后出现CM样病变。 在这个建议中，我们将确定携带体细胞GNAQ R183 Q等位基因的细胞类型的广度，以及如何 与相同表型的非突变细胞相比，突变改变了转录谱（Aim 1）。我们将 在小鼠和斑马鱼中开发新的动物模型，以阐明导致CM的细胞步骤，并将使用 作为测试候选药物的平台（目标2）。我们将深入询问（ANGPT 2）作为一个 组成型活性突变体Gαq的下游功能性介质（Aim 3）。这些研究将加深我们的 了解Gαq活性如何参与毛细血管形态发生，导致第一个动物模型， CM/SWS，并提供一个平台来测试可以预防或消退CM的药物。发现关于 CM的病理生理学也将帮助我们理解其他血管病变的机制 并提高我们识别防止血管过度生长的新途径的能力（例如，癌症）和 在组织修复或工程中促进血管生长。