POEMS综合征中高水平血管内皮生长因子A的异构体组成及其调控机制

POEMS (Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal gammopathy and Skin changes) syndrome is a complex clinical constellation due to an underlying plasma cell dyscrasia. The pathogenesis of this syndrome is not well understood. Serum vascular endothelial growth factor-A (VEGF-A) level elevates in most patients, and can be used as a biomarker in the diagnosis and response evaluation of POEMS syndrome. However, the exact role of VEGF-A in POEMS syndrome is still unclear, which impedes the further mechanism exploration. In the current study, we aim to validate the potential type of cells producing VEGF-A, namely bone marrow plasma cells and stromal cells, via comparison of their ability to generate VEGF-A in cell culture. It is noteworthy that VEGF-A has multiple isoforms, which are characterized by different receptor-binding and downstream signal-initiating properties, and can give rise to distinct angiogenic patterns. The isoform diversity and its functional implication in POEMS syndrome are obscure. Therefore, we will investigate the composite of various VEGF-A isoforms in serum samples and aforementioned VEGF-A-producing cells, hoping to explain the considerable symptomatic heterogeneities among these patients. In addition, we want to explore how bone marrow microenvironment influences the production of VEGF-A isoforms. Cytokines in the bone marrow, including interleukin-6, tumor necrosis factor-α and transforming growth factor-β, constitute a unique microenvironment for residing cells. More importantly, monoclonal protein in POEMS syndrome is almost always λ light chain with restricted immunoglobulin light chain variable gene usage. These restricted λ chains might interact with VEGF-related protein and play a role in VEGF-A secretion. Based on these, we will stimulate VEGF-A-producing cells with cytokines and monoclonal protein in dose- and time-dependent manners, and assess the changes of VEGF-A isoform production. The potentially involved splicing factors, including SF2, SRp40 and SRp55, will be studied via overexpression and knockdown experiments. These studies not only deepen our understanding of POEMS syndrome pathogenesis, but provide clues for novel therapy.

POEMS综合征是一种高致残率的克隆性浆细胞病，发病机制尚不清楚。我们前期工作发现高水平的血清血管内皮生长因子A (VEGF-A)是POEMS综合征诊断和预后的血清标记物，但是我们对VEGF-A在疾病发病中的意义还知之甚少。本申请项目拟通过测定患者骨髓浆细胞和基质细胞表达及分泌VEGF-A的能力，首先确定出POEMS综合征中VEGF-A的细胞来源。其次，通过测定患者血清中各种VEGF-A异构体水平、以及来源细胞内VEGF-A异构体mRNA和蛋白水平，鉴定出POEMS综合征患者的VEGF-A异构体组成，并研究不同组成与临床异质性之间的相关性。最后，用外源性因子体外刺激来源细胞，测定其分泌VEGF-A异构体的组成变化，并研究外源性刺激对VEGF-A特异性剪接因子功能的影响。最终鉴定出调控VEGF-A异构体生成的外源和内源性因素。本项目的实施将有助进一步加深对POEMS综合征发病机制的理解。

本项目以POEMS综合征患者血清中特异升高的VEGF异构体为研究对象，首先通过测定患者骨髓浆细胞和基质细胞表达及分泌VEGF-A的能力，确定出POEMS综合征中患者骨髓内浆细胞是VEGF-A的细胞来源。经流式检测发现单克隆浆细胞VEGF-A水平与多克隆浆细胞类似，但IL-6水平显著高于后者。结合以上结果提出了假设模型：单克隆浆细胞可能通过旁分泌IL-6刺激多克隆浆细胞增生，并共同分泌VEGF-A。通过测定患者血清中各种VEGF-A异构体水平并研究不同组成与临床异质性之间的相关性，发现血清VEGF-165/VEGF-165b高比例组的腹腔积液、肝脾肿大发生率更高，提示VEGF-165b的异构体可能与拮抗腹腔内血管增生相关。通过全外显子测序方法对10例患者骨髓CD138+细胞及外周血CD138+外显子编码区进行对比分析，发现了患者骨髓浆细胞特异性突变基因15个。在扩大样本的靶向基因测序测序中进行验证，共筛选出42例患者中突变率超过10%的基因5个，其中FMNL2基因在患者骨髓浆细胞中突变率高达42.9%，提示POEMS综合征患者浆细胞可能存在一定的生物学特性，并可进一步对其预后价值及分子机制进行探究。通过非靶向脂质组学检测患者及配对健康人血清，发现患者血清中具有潜在预后价值的脂学标记物，并初步建立基于脂质的预后模型。