Role of claudin-5 and hemodynamics in endothelial permeability and susceptibility to atherosclerosis.

摘要

In large arteries atherosclerosis-susceptible regions are associated with disturbed blood flow (flow separations, oscillatory flow reversals with steep shear gradients, and secondary helical flows). In contrast, athero-protected regions are associated with undisturbed flow (unidirectional pulsatile shear stress without flow reversal). Increased endothelial permeability to macromolecules is a prominent feature in disturbed flow regions and it is considered to contribute to this susceptibility. The main barrier to endothelial paracellular transport is the tight junction (TJ), a group of proteins that seal the intercellular space. However, little is known about the relationship between TJ expression and organization, and the hemodynamics. This dissertation tested the hypotheses that differential TJ molecule expression occurs in region of disturbed flow in vivo and the hemodynamics is an important determinant of TJ-meditated endothelial permeability.;In the first section of this study, the expression levels of key TJ molecules were analyzed in two groups of athero-susceptible/disturbed flow, and athero-resistant/undisturbed flow regions---aortic arch (AA) & descending thoracic aorta (DT), and renal branch (RB) & renal artery (RA)---of normal adult swine. In the first comparison group (AA & DT), of the principal molecular component of endothelial TJs, claudin-5 (CLDN5) was the only molecule significantly downregulated at the protein level by 73% in the athero-susceptible/disturbed flow regions of the AA. Occludin (OCC), zonula occludens-1 (ZO-1) and vascular-endothelial cadherin (VE-cad) were equally expressed between the AA and DT. This observation was further reinforced by weak immunostaining of CLDN5 in the AA and a strong staining in the DT, while no differences were detected by immunostaining in other TJ molecules studied. Further analysis revealed a significant inhibition of CLDN5 transcript level by 32% in the AA compared to the DT.;In the second comparison group (RB & RA), careful tissue sectioning and spatial analysis revealed a weak staining of CLDN5 in the cranial region of the RB, an athero-susceptible region exposed to disturbed flow, while intense staining was observed in adjacent athero-resistant regions. Together with the results from AA & DT, these data demonstrate a strong correlation between areas of disturbed flow and low CLDN5 expression in vivo.;In the second section of this study, a cause-and-effect relationship between the hemodynamic environment and CLDN5 expression was directly tested in vitro. Flow waveforms representing the hemodynamic characteristics of in vivo athero-susceptible and athero-protected regions were constructed and a computerized system to apply these waveforms was built. Three-day exposure of human umbilical endothelial cells (HUVECs) to an athero-susceptible waveform resulted in 59% inhibition of CLDN5 protein expression while OCC, ZO-1 and VE-cad remained unchanged. This in vitro finding suggests a direct role of the hemodynamic environment on CLDN5 regulation.;In a direct functional test of CLDN plasticity in regulating the permeability of the endothelial monolayer, CLND5-specific knockdown using two different siRNA constructs significantly increased the permeability of the monolayer by 5.22 fold (construct 1) and by 3.48 (construct 2) in an in vitro transwell assay. These findings suggest that a reduction in CLDN5 expression, as seen by athero-susceptible waveforms in vitro (Chapter 4) or at athero-susceptible regions in vivo (Chapter 3), is sufficient to significantly increase the permeability of the endothelial monolayer to macromolecules.;Taken together, these findings suggest that an increase in endothelial permeability to macromolecules driven by the low expression of CLDN5 in certain vascular regions exposed to disturbed hemodynamic environments, contributes to the spatial susceptibility to atherosclerosis.