Inflammatory environment and oxidized LDL convert circulating human proangiogenic cells (PACs) into functional antigen presenting cells (APCs). Homo sapiens

The function of human circulating proangiogenic cells (PACs) has been described extensively. However, little focus has been placed on understanding how these cells differ in their functions in the presence of microenvironments mimicking vascular inflammation. We hypothesized that exposure to proinflammatory cytokines or the oxLDL, an autoantigen abundant in advanced atherosclerotic plaques, converts PACs into immune-modulating/proinflammatory cells. Hence, we examined the effect of oxLDL and inflammatory stimuli on their phenotype by use of a functional genomics model based on secretome and whole genome transcriptome profiling. PACs obtained from culturing a PBMC fraction in angiogenic medium were primed with DC differentiation cytokines for 3 days and then exposed to proinflammatory cytokines or oxLDL for 2 days. Under these conditions, PACs converted into APCs (APCcy and APCox, respectively), expressed maturation markers CD80 and CD83, and showed an up-regulation of CD86. APCcy and APCox induced a robust T cell BrdU incorporation. Despite a similar ability to induce lymphocyte proliferation, APCcy and APCox differed for the secretory pathway and mRNA expression. Analysis of the differentially expressed genes identified 4 gene "clusters", showing reciprocal modulation in APCcy vs. APCox justifying, according to functional genomics analyses, a different putative function of the cells in antigen processing. Together, these data show that treatment with inflammatory cytokines or oxLDL converts human PAC phenotype and function into that of APCs with similar lymphocyte-activating ability but distinct maturation degree and paracrine functions. Overall design: PBMC-derived PACs were committed with GM-CSF and IL-4 to a dendritic phenotype, and then exposed to proinflammatory cytokines (IL-1β and TNF-α) or to the oxidized form of LDL (oxLDL). Functional assays and whole genome gene expression profiling were performed both on PACs (n=6) and or resulting cytokine- (APCcy, n=6) and oxLDL-induced APCs (APCox, n=6). RNA was reverse transcribed, labeled, and linearly amplified, and then hybridized to HumanHT-12 v.4 Expression BeadChip microarrays. To estimate technical variability, ≈20% of the samples (n=4) were hybridized in duplicate.

人类循环促血管生成细胞（circulating proangiogenic cells, PACs）的功能已被广泛研究，但目前鲜有研究聚焦于阐明这类细胞在模拟血管炎症的微环境中其功能所存在的差异。本研究提出假说：促炎细胞因子或氧化低密度脂蛋白（oxidized low-density lipoprotein, oxLDL，一种在晚期动脉粥样硬化斑块中大量存在的自身抗原）的暴露，可将PACs转化为免疫调节/促炎细胞。因此，本研究基于分泌组学与全基因组转录组分析构建功能基因组学模型，以此探究oxLDL与炎症刺激对PACs表型的影响。
本研究将从外周血单个核细胞（peripheral blood mononuclear cell, PBMC）组分在促血管生成培养基中培养得到的PACs，用树突状细胞（dendritic cell, DC）分化细胞因子诱导3天，随后将其置于促炎细胞因子或oxLDL环境中培养2天。在此培养条件下，PACs可转化为抗原呈递细胞（antigen-presenting cell, APCs，分别记为APCcy与APCox），表达成熟标志物CD80与CD83，并出现CD86的上调。APCcy与APCox可显著诱导T细胞摄取溴脱氧尿苷（bromodeoxyuridine, BrdU）。尽管二者诱导淋巴细胞增殖的能力相近，但APCcy与APCox在分泌通路与mRNA表达层面存在差异。对差异表达基因的分析共鉴定出4个基因簇，这些基因簇在APCcy与APCox中呈现相反的调控模式；结合功能基因组学分析可知，这两类细胞在抗原加工过程中具有不同的推定功能。
综上，本研究数据表明，经促炎细胞因子或oxLDL处理后，人类PACs的表型与功能可转化为APC样细胞：二者激活淋巴细胞的能力相近，但成熟程度与旁分泌功能存在显著差异。
实验整体设计：将外周血单个核细胞来源的PACs用粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GM-CSF）与白细胞介素4（interleukin-4, IL-4）诱导为树突状细胞表型，随后分别暴露于促炎细胞因子（白细胞介素1β（interleukin-1β, IL-1β）与肿瘤坏死因子α（tumor necrosis factor-α, TNF-α））或氧化型低密度脂蛋白（oxLDL）环境中。本研究对PACs（n=6）、细胞因子诱导的APCcy（n=6）以及oxLDL诱导的APCox（n=6）均开展了功能实验与全基因组基因表达谱分析。将RNA进行反转录、标记与线性扩增后，杂交至HumanHT-12 v.4 Expression BeadChip微阵列。为评估技术变异度，我们对约20%的样本（n=4）进行了重复杂交。