ALBERT

Description: Leukocyte transendothelial migration is crucial for innate immunity and inflammation. Upon tissue damage or infection, leukocytes exit blood vessels by adhering to and probing vascular endothelial cells (VECs), breaching endothelial cell-cell junctions, and transmigrating across the endothelium. Transendothelial migration is a critical rate-limiting step in this process. Thus, leukocytes must quickly identify the most efficient route through VEC monolayers to facilitate a prompt innate immune response. Biomechanics play a decisive role in transendothelial migration, which involves intimate physical contact and force transmission between the leukocytes and the VECs. While quantifying these forces is still challenging, recent advances in imaging, microfabrication, and computation now make it possible to study how cellular forces regulate VEC monolayer integrity, enable efficient pathfinding, and drive leukocyte transmigration. Here we review these recent advances, paying particular attention to leukocyte adhesion to the VEC monolayer, leukocyte probing of endothelial barrier gaps, and transmigration itself. To offer a practical perspective, we will discuss the current views on how biomechanics govern these processes and the force microscopy technologies that have enabled their quantitative analysis, thus contributing to an improved understanding of leukocyte migration in inflammatory diseases.

Description: The homeostasis of vascular endothelium is crucial for cardiovascular health and endothelial cell (EC) aging and dysfunction could negatively impact vascular function. Leveraging transcriptome profiles from ECs subjected to various stimuli, including time-series data obtained from ECs under physiological pulsatile flow vs. pathophysiological oscillatory flow, we performed principal component analysis (PCA) to identify key genes contributing to divergent transcriptional states of ECs. Through bioinformatics analysis, we identified that a long non-coding RNA (lncRNA) RAMP2-AS1 encoded on the antisense of RAMP2, a determinant of endothelial homeostasis and vascular integrity, is a novel regulator essential for EC homeostasis and function. Knockdown of RAMP2-AS1 suppressed RAMP2 expression and caused EC functional changes promoting aging, including impaired angiogenesis and increased senescence. Our study demonstrates an integrative approach to quantifying EC aging based on transcriptome changes, which also identified a number of novel regulators, including protein-coding genes and many lncRNAs involved EC functional modulation, exemplified by RAMP2-AS1.

Description: MicroRNAs (miRs) and bone morphogenetic protein receptor–specific Smads are mechano-responsive molecules that play vital roles in modulating endothelial cell (EC) functions in response to blood flow. However, the roles of interplay between these molecules in modulating EC functions under flows remain unclear. We elucidated the regulatory roles of the interplay between miR-487a and Smad5 in EC proliferation in response to different flow patterns. Microarray and quantitative RT-PCR showed that disturbed flow with low and oscillatory shear stress (OS, 0.5 ± 4 dynes/cm2) upregulates EC miR-487a in comparison to static controls and pulsatile shear stress (12 ± 4 dynes/cm2). MiR-487a expression was higher in ECs in the inner curvature (OS region) than the outer curvature of the rat aortic arch and thoracic aorta and also elevated in diseased human coronary arteries. MiR-487a expression was promoted by nuclear phospho-Smad5, which bound to primary-miR-487a to facilitate miR-487a processing. Algorithm prediction and luciferase reporter and argonaute 2-immunoprecipitation assays demonstrated that miR-487a binds to 3′UTR of CREB binding protein (CBP) and p53. Knockdown and overexpression of miR-487a decreased and increased, respectively, phospho-Rb and cyclin A expressions through CBP and p53. A BrdU incorporation assay showed that miR-487a enhanced EC proliferation under OS in vitro and in disturbed flow regions of experimentally stenosed rat abdominal aorta in vivo. These results demonstrate that disturbed flow with OS induces EC expression of miR-487a through its enhanced processing by activated-Smad5. MiR-487 inhibits its direct targets CBP and p53 to induce EC cycle progression and proliferation. Our findings suggest that EC miR-487 may serve as an important molecular target for intervention against disturbed flow–associated vascular disorders resulting from atherosclerosis.

Description: The transition of flow microenvironments from veins to arteries in vein graft surgery induces “peel-off” of venous endothelial cells (vECs) and results in restenosis. Recently, arterial laminar shear stress (ALS) and oscillatory shear stress (OS) have been shown to affect the cell cycle and inflammation through epigenetic controls such as histone deacetylation by histone deacetylases (HDACs) and trimethylation on lysine 9 of histone 3 (H3K9me3) in arterial ECs. However, the roles of H3K9me3 and HDAC in vEC damage under ALS are not known. We hypothesized that the different responses of HDACs and H3K9me3 might cause vEC damage under the transition of venous flow to arterial flow. We found that arterial ECs showed high expression of H3K9me3 protein and were retained in the G0 phase of the cell cycle after being subjected to ALS. vECs became round under ALS with a decrease in the expression of H3K9me3, HDAC3, and HDAC5, and an increase in the expression of vascular cell adhesion molecule 1 (VCAM-1). Inhibition of HDACs activity by a specific inhibitor, phenylbutyrate, in arterial ECs caused similar ALS-induced inflammation and cell loss as observed in vECs. Activation of HDACs and H3K9me3 by ITSA-1, an HDAC activator, could prevent ALS-induced peel-off and reduced VCAM-1 expression in vECs. Moreover, shear stress modulates EC morphology by the regulation of focal adhesion kinase (FAK) expression. ITSA-1 or EGF could increase phosphorylated (p)-FAK expression in vECs under ALS. We found that perturbation of the activity of p-FAK and increase in p-FAK expression restored ALS-induced H3K9me3 expression in vECs. Hence, the abnormal mechanoresponses of H3K9me3 and HDAC in vECs after being subjected to ALS could be reversed by ITSA-1 or EGF treatment: this offers a strategy to prevent vein graft failure.