TLR Accessory Molecule RP105 (CD180) Is Involved in Post-Interventional Vascular Remodeling and Soluble RP105 Modulates Neointima Formation

BackgroundRP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown.Methods and ResultsTLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105−/− mice resulting in a higher proliferation rate. In RP105−/− mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982±974 µm2 vs.1947±278 µm2,p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105−/− mice this effect was more pronounced (10316±1243 µm2 vs.4208±555 µm2,p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500±573 vs.6581±1894 µm2,pConclusionRP105 is a potent inhibitor of post-interventional neointima formation.

**背景**：RP105（CD180）是TLR4的同源蛋白，其缺失胞内TLR4信号结构域，可作为TLR辅助分子及TLR4信号通路的生理性抑制剂。目前RP105在血管重塑，尤其是介入术后血管重塑中的作用尚不明确。
**方法与结果**：通过实时荧光定量PCR（qPCR）和免疫组化检测发现，TLR4与RP105在血管平滑肌细胞（vascular smooth muscle cells, VSMC）及小鼠股动脉段的中膜均有表达。进一步实验显示，RP105基因敲除（RP105−/−）小鼠的VSMC对TLR4配体脂多糖（lipopolysaccharide, LPS）的应答更强，细胞增殖速率更高。与野生型（wild type, WT）小鼠相比，RP105−/−小鼠行股动脉套扎术后的新内膜形成面积显著升高（4982±974 µm² vs. 1947±278 µm²，p=0.0014）。局部给予LPS可在两组小鼠中均增强新内膜形成，但RP105−/−小鼠的该效应更为显著（10316±1243 µm² vs. 4208±555 µm²，p=0.0002），这提示RP105具有功能性调控作用。为开展额外的功能研究，我们表达并纯化了带有或不带有衔接蛋白MD1的小鼠RP105胞外结构域。尺寸排阻色谱-多角度激光光散射（size-exclusion chromatography-multiangle light scattering, SEC-MALS）分析显示，RP105-MD1复合物以功能性2:2同源二聚体形式存在。该蛋白复合物可在离体全血实验中阻断TLR4信号应答。随后我们通过体内基因转染，将编码RP105胞外段及其衔接蛋白MD1的质粒载体导入小鼠体内，以稳定产生内源性可溶性蛋白。可溶性RP105-MD1的表达可显著降低高胆固醇血症小鼠的新内膜形成面积（2500±573 vs. 6581±1894 µm²，p
**结论**：RP105是介入术后新内膜形成的强效抑制剂。