AGE-DEPENDENT PRO-INFLAMMATORY ALTERATIONS IN AORTIC AND MICROVASCULAR ENDOTHELIUM IN HYPERLIPIDEMIC MICE

Abstract

Highlights By 12 months of age, compared with 3 months, male ApoE −/− mice demonstrate increased expression of the pro-inflammatory cell adhesion molecules ICAM1/CD54 and SELE/CD62E, inflammasome-associated cytosolic protein NLRP3, and endothelial-to-mesenchymal transition transcription factor Twist1 in the aortic and microvascular endothelial cells. By 12 months of age, compared with 3 months, male ApoE −/− mice also exhibit increased expression of glycocalyx components (syndecan-1, syndecan-2, syndecan-4, and thrombomodulin) and basement membrane components (laminin, fibronectin, and nidogen-1) in the aortic and microvascular endothelial cells. By 12 months of age, compared with 3 months, male ApoE −/− mice show decreased expression of the atheroprotective transcription factor NRF2 in the endothelium of the aortic intima, suggesting the need in specific therapies for endothelial protection.   Aim. To perform an immunohistochemical analysis of pro-inflammatory activation markers in the aortic endothelial cells and microvascular endothelial cells in male hyperlipidemic ( ApoE −/− ) mice of ascending age. Methods. Cryosections of the aorta from ApoE −/− mice aged 3, 6, 9, and 12 months (n = 6 per time point) were stained with antibodies against pro-inflammatory cell adhesion molecules (VCAM1/CD106, ICAM1/CD54, SELE/CD62E), pro-inflammatory transcription factor NF-κB, inflammasome-associated cytosolic protein NLRP3, endothelial-to-mesenchymal transition transcription factors (Snail, Slug, TWIST1, and ZEB1), endothelial phenotype receptor markers (PECAM1/CD31, KDR/CD309, CDH5/CD144, MCAM/CD146, ENG/CD105, BSG/CD147, Tie2/CD202b, EFNB2, EPHB4), endothelial basement membrane components (laminin, perlecan, fibronectin, type IV collagen, nidogen-1, and nidogen-2), endothelial glycocalyx components (endocan, syndecan-1, syndecan-2, syndecan-4, thrombomodulin, and thrombospondin-1), mechanosensitive transcription factors (KLF2, KLF4, NRF2), and complement regulatory proteins (MAC-IP/CD59, DAF/CD55, MCP/CD46), followed by digital slide acquisition. Semi-quantitative analysis of protein expression in aortic and microvascular endothelium was performed using ImageJ software. Results. By 12 months of age, compared with 3 months, aortic endothelial cells and microvascular endothelial cells of male ApoE −/− mice demonstrated: 1) increased expression of pro-inflammatory cell adhesion molecules ICAM1/CD54 and SELE/CD62E, inflammasome-associated cytosolic protein NLRP3, and endothelial-to-mesenchymal transition transcription factor Twist1, collectively indicating sustained pro-inflammatory activation of the endothelium; 2) increased expression of glycocalyx components (syndecan-1, syndecan-2, syndecan-4, and thrombomodulin), basement membrane components (laminin, fibronectin, and nidogen-1), and complement regulatory protein MAC-IP/CD59, suggesting compensatory activation of synthetic processes; 3) decreased expression of the atheroprotective transcription factor NRF2. No age-related trends were observed in the expression of endothelial phenotype receptor markers or other atheroprotective transcription factors. Conclusion. Dysfunctional aortic and microvascular endothelial cells in 12-month-old male ApoE −/− mice are characterized by pro-inflammatory activation, compensatory synthesis of glycocalyx and basement membrane proteoglycans, and reduced expression of the atheroprotective transcription factor NRF2.