Studies in the Pathogenesis of Systemic Capillary Leak Syndrome

在SCLS患者中，内皮通透性升高和血浆外渗伴随着全身无力、疲劳和肌痛的典型先兆。最典型的体征是低血压、血液浓缩和低蛋白血症三联征。在严重的情况下，患者可能出现四肢全身性水肿、胸膜和心包积液、喉部浮肿并伴有呼吸道阻塞，甚至黄斑浮肿导致失明。 由于临床病例的罕见，对SCLS病因学的研究非常有限。免疫失调可能在疾病的发病机制中起一定作用。虽然对SCLS皮肤的尸检发现真皮内皮细胞周围的基底膜增厚和CD8+CD25+淋巴细胞的渗透，但在光镜和电子显微镜下，其他几名SCLS患者的皮肤活检组织学上是正常的。大多数血清分析结果在SCLS患者的正常范围内，包括补体和C1酯酶抑制物水平。尽管如此，一个流通因素可能会在引发攻击方面发挥作用。1960年，Clarkson报告说，在一次发作期间输注一名SCLS患者的血浆会导致大鼠低血压。然而，在1977年进行的后续研究中，将缓解期或急性发作期间的患者血清或血浆注入豚鼠皮肤或患者自身皮肤未能引起毛细血管通透性。SCLS中存在的单克隆性副蛋白是否直接参与了疾病的发病机制也尚不清楚。对几名患者的皮肤活检进行间接免疫荧光检查，未发现内皮细胞基底膜上的免疫球蛋白复合体。在随后的研究中，纯化的4例SCLS患者的单克隆性副蛋白不能与培养的内皮细胞结合，也不能诱导细胞毒性。最近，有研究表明，健康对照组培养的微血管内皮细胞暴露于SCLS血清中可诱导细胞凋亡，这与活性氧的增加有关。 对于SCLS的急性发作，目前还没有明显有效的治疗方法。皮质类固醇、静脉注射免疫球蛋白(IVIg)、血浆置换和其他免疫抑制剂的使用效果各不相同。也没有有效的维持疗法来防止泄漏事件的发生。坊间和小型病例系列报道表明，磷酸二酯酶抑制剂如茶碱与β-肾上腺素能激动剂(特布他林)联合使用可能会减少某些患者的发作频率和严重程度。然而，这些药物的长期使用可能会受到交感神经副作用的限制。有趣的是，几名MGUS演变为多发性骨髓瘤的SCLS患者在浆细胞恶性肿瘤治疗后渗漏症状减轻，提示浆细胞异常在疾病发病机制中的作用。 我们正在研究单克隆性副蛋白通过免疫(自身免疫或抗独特型)机制触发急性毛细血管渗漏的可能性。抗独特型反应在MGUS中很常见。一种可能性是，副蛋白可能激活淋巴细胞或其他细胞分泌血管内皮生长因子或血管生成素2等通透性因子。高水平的血浆血管内皮生长因子被认为与另一种MGUS相关疾病(POEMS)的病理有关，循环中高水平的血管紧张素2被认为与细胞因子诱导的血管泄漏有关。或者，通透性诱导因子(S)的来源可能是获得性浆细胞或B细胞克隆性群体。这些假说最初将通过纯化单克隆性副蛋白和刺激外周血单核细胞来检验。我们将首先通过流式细胞术检测细胞增殖、表面标志物的表达，以及细胞因子的产生或基因表达。 为了进一步确定与SCLS相关的潜在遗传异常，我们将首先进行细胞遗传学和SNP微阵列分析。这些阵列将通过搜索杂合性缺失(LOH)和拷贝数变异等实体来确定是否存在宏观遗传异常或基因组热点。一旦我们收集了几名SCLS患者的样本，我们还将使用外周血单个核细胞RNA进行基因表达微阵列。基线患者样本将与SCLS发作期间获得的样本以及年龄匹配的健康对照组进行比较。 最后，我们将确定SCLS患者和健康对照组的血管生长和成熟内皮细胞功能的比较。通过表面标记分析可以在外周血中检测到两种不同的循环内皮细胞(CECs)：1)从微血管系统脱落的成熟内皮细胞(CD34+/#8722；CD31+VEGFR2+)和2)骨髓来源的内皮祖细胞(CD34+CD133+VEGFR2+)。与杜德克博士合作，我们将计数SCLS患者外周血中的内皮祖细胞，并通过从内皮祖细胞中提取血液生长内皮细胞(BOEC)来评估分化程度更高的细胞的功能。我们将评估它们的增殖能力，并测量其对血清和/或特定因素的通透性和凋亡反应。我们还将使用这些细胞来评估内皮信号通路和基因表达模式在SCLS和健康患者中的不同之处。最终，这些研究可能会确定SCLS的新治疗靶点，而BOEC将提供一个平台来测试各种化合物预防内皮屏障功能障碍的能力。

In SCLS patients, heightened endothelial permeability and plasma extravasation follows a typical prodrome of generalized weakness, fatigue, and myalgias. The most typical presenting signs are the triad of hypotension, hemoconcentration, and hypoalbuminemia. In severe cases, patients may develop generalized edema of the extremities, pleural and pericardial effusions, laryngeal edema with airway obstruction, and even macular edema leading to blindness. Research studies into SCLS etiology are extremely limited due to the rarity of clinical cases. Immune dysregulation may have a function in disease pathogenesis. Although one postmortem examination of SCLS skin identified basement membrane thickening and infiltration of CD8+CD25+ lymphocytes surrounding endothelial cells in the dermis, skin biopsies taken from several other SCLS patients were histologically normal by light and electron microscopy. Most serum analytes are within the normal range in SCLS patients including complement and C1 esterase inhibitor levels. Nonetheless, a circulating factor could play a role in eliciting attacks. In 1960, Clarkson reported that infusion of plasma from an SCLS patient taken during an episode induced hypotension in rats. However, in a subsequent study done in 1977, injection of patient serum or plasma obtained either during remission or from an acute episode into guinea pig skin or the patients own skin failed to evoke capillary permeability.Whether the monoclonal paraproteins present in SCLS contribute directly to disease pathogenesis is also unclear. Indirect immunofluoresence performed on a skin biopsy from several patients did not reveal IgG complexes on endothelial basement membranes. In a subsequent study, purified monoclonal paraprotein from 4 SCLS patients failed to bind cultured endothelial cells or induce cytotoxicity. Most recently, it was shown that exposure of healthy control cultured microvascular endothelial cells to SCLS serum induced apoptosis, which was associated with increased quantities of reactive oxygen species. There is no clearly effective therapy for acute episodes of SCLS. Corticosteroids, intravenous immunoglobulin (IV Ig), plasmapheresis, and other immunosuppressive agents have been used with variable success. Nor is there efficacious maintenance therapy to prevent leak episodes from occurring. Anecdotal and small case series reports suggest that phosphodiesterase inhibitors such as theophylline in combination with beta-adrenergic agonists (terbutaline) may reduce episode frequency and severity in some patients. However, the chronic use of these drugs may be limited by sympathomimetic side effects. Interestingly, several patients with SCLS in which the MGUS evolved into multiple myeloma saw abatement of their leak symptoms after treatment of the plasma cell malignancy, suggesting a role of the plasma cell dyscrasia in disease pathogenesis. We are investigating the possibility that the monoclonal paraprotein triggers acute capillary leak by an immune (autoimmune or anti-idiotypic) mechanism. Anti-idiotype responses are common in MGUS. One possibility is that the paraprotein may activate lymphocytes or other cells to secrete permeability factors such as VEGF or angiopoietin 2. High plasma VEGF levels are thought to contribute to pathology in another MGUS-related disorder (POEMS), and high circulating levels of Ang2 have been linked to cytokine-induced vascular leak. Alternatively, the source of permeability-inducing factor(s) may be the acquired clonal population of plasma cells or B cells. These hypotheses will be tested initially by purification of the monoclonal paraprotein and stimulation of peripheral blood mononuclear cells. We will initially examine proliferation, expression of surface markers by flow cytometry, and cytokine production or gene expression. To further characterize potential genetic abnormalities associated with SCLS, we will first perform cytogenetics and SNP microarray analysis. These arrays will determine whether macroscopic genetic abnormalities or genomic hot spots are present by searching for entities such as loss of heterozygosity (LOH) and copy number variation. Once we accumulate samples from several SCLS patients, we will also perform gene expression microarrays using peripheral blood mononuclear cell RNA. Baseline patient samples will be compared to specimens obtained during an SCLS attack and to healthy, age-matched controls. Finally, we will determine how blood vessel growth and mature endothelial cell function compare in SCLS patients and healthy controls. Two distinct populations of circulating endothelial cells (CECs) can be detected in peripheral blood by surface marker analysis: 1) mature endothelial cells sloughed from microvasculature (CD34+/−CD133−CD31+VEGFR2+) and 2) bone marrow-derived endothelial progenitor cells (EPCs) (CD34+CD133+VEGFR2+). In collaboration with Dr. Dudek, we will enumerate EPCs in peripheral blood from SCLS patients and evaluate the function of the more differentiated cells by deriving blood-outgrowth endothelial cells (BOEC) from EPCs. We will assess their proliferative capacity and measure permeability and apoptotic responses to serum and/or specific factors. We will also use these cells to evaluate how endothelial signaling pathways and gene expression patterns differ in SCLS and healthy patients. Eventually, such studies may identify new therapeutic targets for SCLS, and BOEC will provide a platform to test the ability of various compounds to prevent endothelial barrier dysfunction.