Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Humboldt foundation Background Calcium-Sensing Receptor (CaSR) is a cell surface G-protein coupled receptor that senses calcium in the extracellular environment. This receptor is widely studied in mineral homeostasis due to its presence in varied calcitropic tissues. However, CaSR is also present on the surface of vascular and hematopoietic cells and recent studies suggest that this receptor can also have pathological consequences related to cardiovascular diseases, like atherosclerosis. Since endothelial cells are crucial players in atherogenesis, we aimed to investigate which role endothelial CaSR plays in the development and progression of atherosclerosis. Methods We cross-bred CaSRflox/flox Apoe-/- mice with BmxCreERT2 Apoe-/- (endothelial cell specific Cre driver) mice to obtain tamoxifen-inducible mice which have an endothelial cell specific deletion of CaSR. The mice were then fed with a high fat diet (HFD) for 4 or 12 weeks, reflecting early and late atherosclerosis, respectively. We used histological and immuno-fluorescent stainings to analyse the atherosclerotic lesion size and its cellular and acellular composition. Systemic effects on leukocytes were evaluated using flow-cytometry, while intra-vital microscopy was used to study leukocyte adhesion to activated endothelium in-vivo. Further mechanistic studies were performed in human coronary artery endothelial cells (HCAECs) in-vitro. Results Endothelial CaSR deficiency in mice resulted in significantly smaller lesion size in early atherosclerotic stages (4 weeks HFD), whereas late atherosclerotic lesions (12 weeks HFD) were not affected by the lack of endothelial CaSR. Moreover, the phenotype of atherosclerotic plaques, characterized by macrophage, smooth muscle cell, collagen and necrotic core content remained unaltered between wild-type and endothelial cell specific CaSR knockout mice. Interestingly, leukocyte adhesion in-vivo, especially neutrophil adhesion, was significantly reduced in endothelial cell CaSR deficient mice. In line with this, overexpression of CaSR in HCAECs in-vitro increased the production of inflammatory cytokines and expression of adhesion molecules ICAM and VCAM. Conclusion Our results indicate that while endothelial cell specific CaSR is involved in the induction of atherosclerosis, it does not play a major role in its progression. CaSR has a major influence on endothelium-leukocyte interaction and endothelial inflammation. The increased expression of ICAM/VCAM suggest that CaSR mainly mediates such adhesive effects by modulating integrin expression. Thereby this study highlights that CaSR plays a crucial role in atherosclerosis development, rendering it a novel, so far unexplored therapeutic target.

中文翻译：

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内皮CaSR通过促进细胞粘附和局部炎症参与动脉粥样硬化的诱导

资金致谢 资金来源类型：公共拨款——仅限国家预算。主要资金来源：洪堡基金会背景钙敏感受体 (CaSR) 是一种细胞表面 G 蛋白偶联受体，可在细胞外环境中感应钙。由于该受体存在于不同的钙质组织中，因此在矿物质稳态中被广泛研究。然而，CaSR 也存在于血管和造血细胞的表面，最近的研究表明，这种受体也可能产生与心血管疾病相关的病理后果，如动脉粥样硬化。由于内皮细胞是动脉粥样硬化形成的关键参与者，我们旨在研究内皮 CaSR 在动脉粥样硬化的发展和进展中所起的作用。方法 我们将 CaSRflox/flox Apoe-/- 小鼠与 BmxCreERT2 Apoe-/-（内皮细胞特异性 Cre 驱动器）小鼠杂交，以获得内皮细胞特异性缺失 CaSR 的他莫昔芬诱导型小鼠。然后给小鼠喂食高脂肪饮食 (HFD) 4 或 12 周，分别反映早期和晚期动脉粥样硬化。我们使用组织学和免疫荧光染色来分析动脉粥样硬化病变的大小及其细胞和非细胞成分。使用流式细胞术评估对白细胞的全身影响，而活体显微镜用于研究白细胞在体内与活化的内皮细胞的粘附。在体外对人冠状动脉内皮细胞 (HCAEC) 进行了进一步的机制研究。结果 小鼠内皮 CaSR 缺乏导致早期动脉粥样硬化阶段（4 周 HFD）的病变尺寸显着减小，而晚期动脉粥样硬化病变（12 周 HFD）不受内皮 CaSR 缺乏的影响。此外，以巨噬细胞、平滑肌细胞、胶原蛋白和坏死核心含量为特征的动脉粥样硬化斑块的表型在野生型和内皮细胞特异性 CaSR 敲除小鼠之间保持不变。有趣的是，体内白细胞粘附，尤其是中性粒细胞粘附，在内皮细胞 CaSR 缺陷小鼠中显着降低。与此一致，体外HCAEC中CaSR的过表达增加了炎性细胞因子的产生和粘附分子ICAM和VCAM的表达。结论 我们的结果表明，虽然内皮细胞特异性 CaSR 参与了动脉粥样硬化的诱导，但它在其进展中并不起主要作用。CaSR 对内皮-白细胞相互作用和内皮炎症有重要影响。ICAM/VCAM 的表达增加表明 CaSR 主要通过调节整合素表达来介导这种粘附作用。因此，这项研究强调了 CaSR 在动脉粥样硬化发展中起着至关重要的作用，使其成为迄今为止尚未探索的新型治疗靶点。