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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:10.)
© 2006 American Heart Association, Inc.

Editorials

Tightening the Barrier

Mechanical Forces and the Control of Endothelial Permeability

Jeremy D. Pearson

From King’s College London, Cardiovascular Division, Guy’s Campus, London.

Correspondence to Jeremy D. Pearson, King’s College London, Cardiovascular Division, Guy’s Campus, London SE1 1UL. Email jeremy.pearson{at}kcl.ac.uk

Interendothelial junctional clefts have long been supposed to be the major sites at which water and solutes leave the vessel lumen (except in vessels with fenestrated or discontinuous endothelium) to reach extravascular tissue. Pioneering electron microscopy studies by Palade with the Simionescus demonstrated this phenomenon directly, showing that proteins of a molecular diameter up to &20 Å could permeate these clefts in capillaries, and rather larger proteins could do so in post-capillary venules. They were also the first, by usng freeze fracture techniques, to show that the extent and complexity of the arrangement of interendothelial junctional proteins varied substantially across the vasculature, in a manner consistent with what was known about vessel permeability: the most complex and extensive junctions are found in arteries and in the small vessels forming the blood-brain barrier, whereas the least complex and extensive are found in post-capillary venules.^1–3 However, at that time neither the identity of the major molecular components of the junctions was known nor had anyone begun to consider what mechanisms might lead to this graded variation of endothelial phenotype.

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Nonetheless, the distinctive shapes of endothelial cells in situ according to their position in the vascular tree suggests their ability to respond to physical forces such as shear stress—an insight usually attributed to Virchow, who described the variation in endothelial cell morphology and proposed it more than 150 years ago. Direct confirmation came only in the 1970s from observations of the endothelium in grafted vessels in vivo and in the 1980s from in vitro experiments with cultured endothelial cells, which additionally demonstrated that endothelium responds rapidly to mechanical forces, for example by altering the synthesis of vasoactive mediators including nitric oxide and prostacyclin.⁴ The realization that endothelial physiology is modulated by physical forces, both acutely and in the longer term, led to extensive searches to understand the molecular mechanisms involved in the initial sensing of force and the subsequent transduction pathways. Candidate mechanosensors include ion channels, membrane associated protein kinases or phospholipases, or intracellular signaling pathways activated by cytoskeletal rearrangements.^4,5 By the 1990s several genes had been identified whose endothelial expression was regulated in vitro in response to shear or stretch, with subsequent demonstration of their differential expression in vivo either between arteries and veins or even between the internal and external curvature of a single vessel,⁵ and the more recent use of gene array technology has greatly increased our knowledge of the spectrum of endothelial phenotypic changes induced by increased or disturbed shear stress.⁵

We now know that the major components found in the tight junctional regions linking adjacent endothelial cells include the occludin, claudin, and JAM (junctional adhesion molecule) protein families, linked to the intracellular actin cytoskeleton and to other signal transducing molecules via ZO (zonula occludens) and related proteins.⁶ Although several proximal signaling pathways have been identified (eg, elevation of cytosolic Ca²⁺) leading to acute and transient increases of endothelial permeability in response to inflammatory mediators (such as histamine and thrombin) or growth factors (notably VEGF), the distal signaling pathways are still not well understood, but altered phosphorylation of tight junction proteins has been implicated. Although the pathways may involve direct modulation of the phosphorylation state of occludin or ZO-1, leading to altered partner protein binding and altered cellular distribution, the integrity of the junctions can also be modified indirectly by interference with actin dynamics (eg, by small GTPases such as Rho and Rac, or by cytochalasin D).^6,7 By contrast, almost nothing is known of the longer term regulation of endothelial junctional proteins, for example in response to mechanical forces.

In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Collins et al⁸ present the results of experiments designed to study the regulation of occludin and ZO-1 in bovine aortic endothelial cells in culture when exposed to cyclic stretch of a magnitude similar to that experienced in larger arteries. They report a 2-fold increase in the level of both proteins after 24 hours, together with an increased association of occludin and ZO-1 and increased localization to cell-cell junctions, and a parallel reduction in transendothelial protein permeability. The steady state level of occludin mRNA (but not ZO-1 mRNA) was also increased. The changes in the levels of the proteins were accompanied by alterations in the phosphorylation state of each: decreased tyrosine phosphorylation of occludin and increased serine/threonine phosphorylation of ZO-1. Inhibiting tyrosine phosphatase (with dephostatin) or protein kinase C (with rottlerin) blocked the phosphorylation changes in occludin or ZO-1, respectively, and in either case reduced the association between the two proteins and their localization to the cell border.

These results thus add significantly to our knowledge of how tight junctions (and, by inference, permeability) can be regulated in endothelial cells. However, we are still some way from being able to provide a coherent picture of the effects of physical forces on endothelial cell junctions, and it is not certain that the current results have a direct bearing on understanding the differences in tight junctional complexity found in vivo in different vascular beds. Physical forces alone cannot be invoked to explain the differences, because of the example of the blood-brain barrier—here trophic influences from astrocytes, known to invoke other specific properties of the endothelium in these vessels, are likely to play an important role.⁹ Therefore analogous mechanisms (eg, levels of paracrine growth factors) may be more important than the direct effects of physical forces in determining the complexity of endothelial tight junctions in different parts of the vasculature, even though the latter mechanism seems intrinsically logical and appealing, particularly given the significant number of other phenotypic characteristics of endothelium that are regulated by physical forces.^4,5

Nonetheless, although alterations in the levels of mRNA for a small number of other cell junctional proteins (eg, connexin 37 and PECAM) have been noted in the half dozen published articles using gene arrays to identify phenotypic changes in endothelial cells in response to shear stress, none of these studies noted changes in occludin or claudin (or ZO-1).⁵ As discussed by Collins et al,⁸ two other recent articles have specifically studied occludin protein expression in endothelial cells in response to shear stress, with contrasting results. In one, exposure of cultured bovine aortic endothelial cells to shear stress substantially reduced occludin expression by comparison with static cultures.¹⁰ In the other, where porcine carotid arteries were perfused ex vivo at high (physiological) or low shear stress, occludin expression was lower after exposure to low shear stress.¹¹

At present, the only safe conclusion is the rather unsatisfactory one that hemodynamic or other physical forces acting on endothelial cells can clearly modulate tight junctional protein expression and organization, but the fundamental mechanisms controlling the outcome and their relative importance in vivo remain poorly defined.

How can we move on to get a more complete picture ? One initial approach would be to use promoter-reporter gene constructs to define whether occludin, claudin, or ZO-1 expression are directly regulated by increased shear or stretch and, if so, to identify the promoter binding elements responsible. However, the bulk of the evidence summarized above suggests that cellular localization, organization, and turnover of tight junctional components, probably controlled by phosphorylation/dephosphorylation, are more critical than the level of mRNA transcription or translation. Given the multiple protein species in the tight junctions, each with its own complex multiple phosphorylation sites, finding the key signals may prove difficult. Nonetheless, a recent comprehensive review of tight junction assembly and disassembly, even though it does not directly address the long term regulation of tight junction expression, suggests several pointers.⁷ First, regulation of the ability of claudin to interact correctly with the other protein components seems to be pivotal for tight junction formation and function, so it may be prudent to monitor the expression and localization of claudin as a readout. Second, some of the important signaling pathways regulating the acute increases in permeability caused by inflammatory agonists have been defined: these include activation of PKC isoforms (also implicated in the current study ⁸) and activation of the extracellular signal regulated kinase (ERK)/Ras/Rab pathway. It would therefore seem sensible to manipulate these pathways (by expression of constitutively active or dominant negative mutant proteins) and test their effects on the ability of mechanical forces to modulate tight junction component phosphorylation, expression, and localization, together with effects on permeability. In parallel the effects of mechanical forces on the steady state endogenous expression and activation of these signaling pathways could be determined. In this respect it is worth noting that different steady state patterns of ERK activation (and NF-B activation) in endothelial cells according to exposure to linear or disturbed shear stress have already been reported.^12,13

Defining which mechanisms are important in vivo will be more challenging, because any model in which surgical or pharmacological manipulation is used to alter shear or pulsatility of flow is likely to introduce changes in other parameters, but investigating endothelial junctional remodeling in such studies could provide valuable insights. After all, just over 30 years ago it was the bold and imaginative experiments by Flaherty et al,¹⁴ who reinserted lengths of canine artery in vivo after cutting down one side and sewing them so that the reinserted segment was at right angles to its former orientation, that first demonstrated that the endothelial cells then gradually reoriented to line up again in the direction of blood flow—before similar observations were made in the simpler and more controlled environment of a culture dish.

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