Venous Malformations (VM): A Murine Mdoel to Identify Therapies to Targer Aberrant Venous Development

描述(申请人提供)：静脉畸形(VM)是一种慢性疾病，常见于出生时或儿童时期。VMS是由增宽的、形状不规则的静脉组成的病变。许多区域缺少平滑肌细胞。随着年龄的增长，病变往往会扩大，并可能突然扩大，导致血栓形成而导致畸形和/或疼痛。目前还没有针对性的治疗方法，对VM的治疗也非常有限，仅包括硬化治疗和重建手术。治疗后，皮损经常复发。胚系或体细胞TIE2突变与VM有关。Tie2是一种内皮受体酪氨酸激酶，调节维持血管静止和促进血管生成。我们的假设是，由于血管周围细胞的错误分化和/或募集，内皮细胞中的TIE2突变导致了异常的静脉发育。因此，我们建议使用在患者中发现的表达TIE2突变的内皮细胞来概括小鼠的VM表型。VM的小鼠模型从未被报道过。我们的初步数据表明，表达TIE2-L914F突变受体的人脐静脉内皮细胞，当注射到小鼠皮下时，会形成倾向于随着时间的推移而生长的血管病变。组织学显示血管过度增宽，充满血液。异常通道周围血管周细胞稀少，病变内血流缓慢，以非搏动性为主。作为对照，我们用表达TIE2野生型(WT)的人脐静脉内皮细胞作为对照，在我们的模型中，这些细胞不能形成血管。将正常内皮细胞与骨髓间充质祖细胞(BmMPC)联合注射可形成大小规则的血管。人脐静脉内皮细胞TIE2-L914F与bmMPC联合注射可形成管腔扩大、血管周细胞稀少的血管，bmMPC分化/成熟不明显。人脐静脉内皮细胞TIE2-WT的作用与正常内皮细胞相似。因此，我们假设TIE2突变抑制血管周围细胞的分化，并抑制管腔大小的正确控制。我们的研究将集中在：目的1-建立一个反映患者疾病的VM-小鼠模型，使用带有突变的TIE2受体的EC或从患者VM分离的细胞；目的2-确定TIE2突变对血管周围细胞分化、管腔大小控制和运动的影响，我们将在小鼠VM模型和3D胶原体外模型中使用bmMPC、周细胞或SMCs；目的3-进行临床前研究，以确定一种新的针对VM的靶向治疗方法，以使病理性血管系统正常化或诱导退化，避免疾病的反弹。

DESCRIPTION (provided by applicant): Venous malformation (VM) is a chronic condition often seen at birth or during childhood. VMs are lesions composed by widened, abnormally shaped veins. Smooth muscle cells are absent in many areas. The lesion tends to enlarge with age and can suddenly expand causing deformity and/or pain as a result of clot formation. No targeted therapies are available, and treatments for VM are very limited, including only sclerotherapy and reconstructive surgery. After treatment, lesions often recur. Germline or somatic TIE2 mutations have been associated with VMs. TIE2 is an endothelial receptor tyrosine kinase that regulates both maintenance of vascular quiescence and promotion of angiogenesis. Our hypothesis is that TIE2 mutations in the endothelial cells cause aberrant venous development due to an incorrect differentiation and/or recruitment of perivascular cells. Therefore we propose to use endothelial cells expressing the TIE2 mutations found in patients to recapitulate the VM phenotype in mouse. A murine model of VM has never been reported. Our preliminary data demonstrate that HUVECs expressing TIE2-L914F mutated receptor, when injected subcutaneously in mouse, form vascular lesions that tend to grow over time. The histology reveals excessively widened blood-filled vessels. Perivascular cells around the abnormal channels are scarce in number, and blood flow in the lesion is slow and mainly non-pulsatile. As control we used HUVECs expressing TIE2-Wild Type (WT), these cells are incapable of forming blood vessels in our model. Injection of normal ECs in combination with bone marrow mesenchymal progenitor cells (bmMPC) results in formation of regular sized blood vessels. HUVECs TIE2-L914F injected in combination with bmMPC formed instead vessels with enlarged lumen, with scarce perivascular cells, and no evident differentiation/maturation of bmMPC. HUVECs TIE2-WT behaved similarly to normal ECs. We thereby hypothesize that the TIE2 mutation inhibits perivascular cells differentiation, and the correct control of the lumen siz. Our research will focus on: Aim 1- Establishing a VM-murine model that reflects the patient's disease, with the use of EC with mutated TIE2 receptor or cells isolated from patient VMs; Aim 2- Determine the effects of TIE2 mutations in the perivascular cell differentiation, control of lumen size, and motility we will use bmMPC, pericytes or SMCs, in the murine VM model and in 3D collagen in vitro model; Aim 3- Perform pre-clinical studies to identify a novel targeted treatment for VMs, in order to normalize or induce regression of the pathological vasculature and avoid a rebound of the disease.