Proteomics of endothelial cell secretome after exposure to calciprotein particles

Calciprotein particles (CPPs), assembled as a result of molecular interactions between fetuin-A and nascent calcium phosphate clusters, are indispensable scavengers of excessive Ca2+ and PO43– ions, thus representing an elegant mechanism which regulates mineral homeostasis. Generation of CPPs represents an evolutionary mechanism to prevent blood supersaturation with Ca2+ and PO43– ions (e.g., as a result of bone resorption) and to thwart extraskeletal calcification, a pathological condition which is frequent in patients with chronic kidney disease. It is believed that formation of CPPs accompanied evolution from the appearance of bony fish; the existence of CPPs in more ancient animals remains uncertain. Shortly after emergence, amorphous, spherical, and submicrometer-sized (30-100 nm) primary CPPs (CPP-P) evolve into crystalline, spindle- or needle-shaped, and micrometer-sized (100-300 nm) secondary CPPs (CPP-S). Upon executing their function of aggregating Ca2+ and PO43– ions, CPPs are removed from the circulation by endothelial cells (ECs), monocytes, and liver or spleen macrophages. Internalisation and digestion of CPPs by ECs induce a chain of detrimental events including an increase in cytosolic Ca2+, mitochondrial and endoplasmic reticulum stress, nuclear factor (NF)-κB-mediated transcriptional response and cytokine release, ultimately contributing to the development of pro-inflammatory endothelial activation, chronic low-grade inflammation, and inflammaging. Pathological permeability of dysfunctional endothelium promotes lipid retention and leukocyte extravasation, together contributing to the development of vascular inflammation, proteolytic environment, degradation of basement membrane and internal elastic lamina, and contractile-to-synthetic switch of vascular smooth muscle cells. Such an ensemble of molecular events ultimately induces intimal hyperplasia, vascular remodeling, and atherosclerotic progression. Hence, CPPs act as a double-edged sword which rescues the human body from an acute life-threatening disease (i.e., extraskeletal calcification) at the cost of promoting long-lasting conditions (i.e., endothelial dysfunction and atherosclerosis). Among the several forms of calcium transfer (free Ca2+ ions, colloidal calciprotein monomers, and particulate CPPs), CPPs are the most efficient in delivering calcium stress to ECs because of their deposition and dissolution in lysosomes, followed by a lysosomal membrane permeabilisation and an excessive Ca2+ migration into the cytosol. The molecular pattern of CPP-induced calcium stress within the ECs is well defined and includes elevated expression of cell adhesion molecules (VCAM1, ICAM1, and E-selectin) and endothelial-to-mesenchymal transition transcription factors (SNAI1, SNAI2, TWIST1, and ZEB1) in combination with an endothelial nitric oxide synthase (eNOS) uncoupling. However, our knowledge on extracellular signatures of such endothelial response remains limited to an augmented release of pro-inflammatory cytokines (IL-6, IL-8, MCP-1/CCL2, MIF, CXCL1, and MIP-3α/CCL20) and pro- or anti-thrombotic molecules (serpin E1/PAI-1 and uPAR). As EC-secreted bioactive factors (termed angiokines) control vascular tone, maintain hemostasis, and regulate instructive signaling governing local homeostasis within the most organs, decryption and interpretation of endothelial secretome in physiological and pathological conditions is of utmost importance. Discovery of a circulating biomarkers specific for endothelial dysfunction, which can be measured by a routine enzyme-linked immunosorbent assay, might inform on specific disease states and prompt to the respective pharmacological interventions. Here, we aimed at deciphering the secretome of ECs treated with either CPP-P or CPP-S, employing ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) for the analysis of serum-free cell culture supernatant. For the objective comparison of heterogeneous arterial ECs, we utilised human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) which comprise an innermost lining in atheroprone and atheroresistant blood vessels (coronary artery and internal thoracic artery, respectively).

钙蛋白颗粒（Calciprotein particles, CPPs）是由胎球蛋白A与新生磷酸钙团簇通过分子相互作用组装形成的颗粒，是清除体内过量Ca²+和PO₄³-离子的不可或缺的清道夫，代表了调控矿物质稳态的精妙机制。钙蛋白颗粒的生成是一种进化形成的机制，可防止血液中Ca²+和PO₄³-离子过饱和（例如因骨吸收所致），并阻止骨外钙化——这是慢性肾脏病患者中常见的病理状态。学界普遍认为，钙蛋白颗粒的形成伴随着硬骨鱼出现后的进化过程，而更古老动物体内是否存在钙蛋白颗粒仍不明确。钙蛋白颗粒诞生后不久，无定形、球形、亚微米级（30~100 nm）的初级钙蛋白颗粒（CPP-P）会演变为结晶态、纺锤形或针形、微米级（100~300 nm）的次级钙蛋白颗粒（CPP-S）。在完成聚集Ca²+和PO₄³-离子的功能后，钙蛋白颗粒会被内皮细胞（endothelial cells, ECs）、单核细胞以及肝脏或脾脏巨噬细胞从循环系统中清除。内皮细胞对钙蛋白颗粒的内化与消化会引发一系列有害事件，包括胞质钙离子浓度升高、线粒体与内质网应激、核因子κB（NF-κB）介导的转录应答以及细胞因子释放，最终促成促炎性内皮活化、慢性低度炎症与炎性衰老的发生。功能失调的内皮细胞出现病理性通透性升高，会促进脂质滞留与白细胞外渗，共同推动血管炎症、蛋白水解微环境形成、基底膜与内弹性膜降解，以及血管平滑肌细胞从收缩型表型向合成型表型转换。这一系列分子事件最终会引发内膜增生、血管重构与动脉粥样硬化进展。因此，钙蛋白颗粒堪称一把双刃剑：它能帮助机体规避急性致命性疾病（即骨外钙化），却以促进长期病症（即内皮功能失调与动脉粥样硬化）为代价。在多种钙转运形式（游离Ca²+离子、胶体钙蛋白单体以及颗粒型钙蛋白颗粒）中，钙蛋白颗粒是向内皮细胞传递钙应激最有效的形式——这是因为它们会在溶酶体内沉积并溶解，随后引发溶酶体膜通透性改变，使过量钙离子迁移至胞质中。内皮细胞内钙蛋白颗粒诱导的钙应激分子模式已得到明确界定，包括细胞黏附分子（VCAM1、ICAM1与E-选择素）以及内皮细胞向间充质细胞转化转录因子（SNAI1、SNAI2、TWIST1与ZEB1）的表达上调，同时伴随内皮一氧化氮合酶（eNOS）解偶联。不过，目前学界对于此类内皮应答的细胞外特征的认知，仅局限于促炎细胞因子（IL-6、IL-8、MCP-1/CCL2、MIF、CXCL1与MIP-3α/CCL20）以及促凝/抗凝分子（丝氨酸蛋白酶抑制蛋白E1/纤溶酶原激活物抑制剂1（serpin E1/PAI-1）与尿激酶型纤溶酶原激活物受体（uPAR））的释放增加。由于内皮细胞分泌的生物活性因子（称为血管细胞因子）可调控血管张力、维持血液凝固稳态，并调节支配多数器官局部稳态的指导性信号，解析生理与病理状态下的内皮细胞分泌组具有至关重要的意义。发现可通过常规酶联免疫吸附试验检测的、特异性针对内皮功能失调的循环生物标志物，或可辅助判断特定疾病状态，并指导相应的药物干预。本研究旨在解析经初级钙蛋白颗粒或次级钙蛋白颗粒处理的内皮细胞的分泌组，采用超高效液相色谱-串联质谱（UHPLC-MS/MS）分析无血清细胞培养上清液。为了对异质性动脉内皮细胞进行客观比较，我们选用了人冠状动脉内皮细胞（HCAEC）与人胸廓内动脉内皮细胞（HITAEC）——它们分别是易动脉粥样硬化血管（冠状动脉）与抗动脉粥样硬化血管（胸廓内动脉）的内层衬里细胞。