Taggi Valerio, Schäfer Anima M, Kinzi Jonny, Ritz Danilo, Seibert Isabell, Oswald Stefan, Zu Schwabedissen Henriette E Meyer
Biopharmacy, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland.
Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland.
Mol Neurobiol. 2025 Mar 14. doi: 10.1007/s12035-025-04807-7.
The blood-brain barrier (BBB) is essential for central nervous system (CNS) homeostasis by regulating permeability between the bloodstream and brain. This study evaluates the immortalized human brain capillary endothelial cell lines hCMEC/D3 and hBMEC for their use as a brain endothelial cells to investigate the OATP2B1 transporter following adenoviral infection. We assessed the impact of adenoviral-mediated OATP2B1 expression on BBB marker proteins and transporters using targeted and untargeted mass spectrometry-based proteomics. Targeted proteomics identified measurable levels of endothelial markers PECAM1 and CDH5 in hCMEC/D3, whereas these markers were undetectable in hBMEC. Both cell lines exhibited similar Pgp levels, while BCRP was absent in hCMEC/D3. The expression of uptake transporters was also evaluated, revealing comparable levels of GLUT1, ENT1, MCT1 and OAT7 in hCMEC/D3 and hBMEC. Although OATP2B1 levels did not significantly increase post-infection in targeted proteomics, untargeted proteomics confirmed enhanced OATP2B1 expression. Other BBB markers and transporters remained unaffected in both cell lines. Notably, hCMEC/D3 demonstrated a stronger BBB phenotype, indicated by higher expression of BBB markers and transporters, while adenoviral infection was more effective in hBMEC. The differences between targeted and untargeted proteomics underscore the need for diverse methods to verify protein expression levels. This comparative analysis provides insights into the strengths and limitations of hCMEC/D3 and hBMEC for BBB research, particularly regarding drug transport mechanisms.
血脑屏障（BBB）通过调节血液与大脑之间的通透性，对中枢神经系统（CNS）的稳态至关重要。本研究评估了永生化的人脑血管内皮细胞系hCMEC/D3和hBMEC作为脑内皮细胞在腺病毒感染后研究OATP2B1转运蛋白的用途。我们使用基于靶向和非靶向质谱的蛋白质组学评估了腺病毒介导的OATP2B1表达对BBB标记蛋白和转运蛋白的影响。靶向蛋白质组学在hCMEC/D3中鉴定出可测量水平的内皮标记物PECAM1和CDH5，而在hBMEC中未检测到这些标记物。两种细胞系的Pgp水平相似，而hCMEC/D3中不存在BCRP。还评估了摄取转运蛋白的表达，结果显示hCMEC/D3和hBMEC中GLUT1、ENT1、MCT1和OAT7的水平相当。尽管在靶向蛋白质组学中，感染后OATP2B1水平没有显著增加，但非靶向蛋白质组学证实了OATP2B1表达增强。两种细胞系中的其他BBB标记物和转运蛋白均未受到影响。值得注意的是，hCMEC/D3表现出更强的BBB表型，表现为BBB标记物和转运蛋白的表达更高，而腺病毒感染在hBMEC中更有效。靶向和非靶向蛋白质组学之间的差异强调了需要多种方法来验证蛋白质表达水平。这种比较分析为hCMEC/D3和hBMEC在BBB研究中的优势和局限性提供了见解，特别是在药物转运机制方面。
