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Microvascular barrier dysfunction is implicated in the initiation and progression of inflammation, posttraumatic complications, sepsis, ischaemia-reperfusion injury, atherosclerosis, and diabetes. Under physiological conditions, a precise equilibrium between endothelial cell-cell adhesion and actin-myosin-based centripetal tension tightly controls the semi-permeability of microvascular barriers. Myosin light chain kinase (MLCK) plays an important role in maintaining the equilibrium by phosphorylating myosin light chain (MLC), thereby inducing actomyosin contractility and weakening endothelial cell-cell adhesion. MLCK is activated by numerous physiological factors and inflammatory or angiogenic mediators, causing vascular hyperpermeability. In this review, we discuss experimental evidence supporting the crucial role of MLCK in the hyperpermeability response to key cell signalling events during inflammation. At the cellular level, in vitro studies of cultured endothelial monolayers treated with MLCK inhibitors or transfected with specific inhibiting peptides have demonstrated that induction of endothelial MLCK activity is necessary for hyperpermeability. Ex vivo studies of live microvessels, enabled by development of the isolated, perfused venule method, support the importance of MLCK in endothelial permeability regulation in an environment that more closely resembles in vivo tissues. Finally, the role of MLCK in vascular hyperpermeability has been confirmed with in vivo studies of animal disease models and the use of transgenic MLCK210 knockout mice. These approaches provide a more complete view of the role of MLCK in vascular barrier dysfunction.

In response to infections or tissue injury, circulating leucocytes adhere to and migrate from the vessel lumen to interstitial inflammatory sites to combat invading pathogens. However, these defensive actions may also cause host tissue injury and microvascular dysfunction through oxidative bursts or enzyme release. For decades, the interaction between leucocytes and microvessel walls has been considered as a critical event leading to organ dysfunction. Extensive investigations have therefore focused on blocking specific adhesive ligands to prevent tissue injury. However, anti-adhesion therapies have shown limited success in preventing vascular dysfunction in clinical trials. Numerous studies have demonstrated temporal and spatial dissociations of leucocyte adhesion and/or emigration from permeability increases. The mechanisms that initiate the adhesion cascade have been found to be distinct from those that trigger the leucocyte oxidative burst responsible for increasing microvessel permeability. Recent studies demonstrated that endothelial activation by inflammatory mediators is critical for initiating platelet adhesion and platelet-dependent leucocyte recruitment resulting in augmented increases in microvessel permeability. These new developments suggest that targeting endothelial activation via directly enhancing endothelial barrier function might be a more efficient strategy than focusing on anti-adhesion or platelet/leucocyte depletion to prevent vascular damage during inflammation. Owing to space limitations and the wide range of studies in the field, this article will not serve as a comprehensive review. Instead, it will highlight the emerging evidence of adhesion-uncoupled permeability changes and establish a basis for re-evaluating the coupled relationship between leucocyte/platelet activation and microvessel permeability to achieve a better understanding of permeability regulation during inflammation.

Multiple processes modulate net blood-to-tissue exchange in a microvascular unit in normal and pathophysiological conditions. These include mechanisms that control the number and type of microvessels perfused, the balance of adhesion and contractile forces that determine the conductance of the spaces between endothelial cells to water and solutes, the pressure and chemical potential gradients determining the driving forces through these conductive pathways, and the organization of barriers to macromolecules in the endothelial glycocalyx. Powerful methods are available to investigate these mechanisms at the levels of cultured endothelial monolayers, isolated microvessels, and the microvascular units within intact organs. Here we focus on current problems that limit the integration of our knowledge of mechanisms investigated in detail at the cellular level into a more complete understanding of modulation of blood-to-tissue exchange in whole organs when the endothelial barrier is exposed to acute and more long-term inflammatory conditions. First, we review updated methods, applicable in mouse models of vascular permeability regulation, to investigate both acute and long-term changes in permeability. Methods to distinguish tracer accumulation due to change in perfusion from real increases in extravascular accumulation are emphasized. The second part of the review compares normal and increased permeability in individually perfused venular microvessels and endothelial cell monolayers. The heterogeneity of endothelial cell phenotypes in the baseline state and after exposure to injury and inflammatory conditions is emphasized. Lastly, we review new approaches to investigation of the glycocalyx barrier properties in cultured endothelial monolayers and in whole-body investigations.

Ischaemia and reperfusion (I/R) elicits an acute inflammatory response that is characterized by the recruitment of inflammatory cells, oxidative stress, and endothelial barrier failure. Over the past three decades, much progress has been made in our understanding of the mechanisms that underlie the inflammatory response and microvascular dysfunction associated with I/R. This review is focused on the role of leucocytes (neutrophils and T-lymphocytes) and platelets, and their activation products, as mediators of I/R-induced endothelial barrier failure. The contributions of cytokines, chemokines, and oxidative stress to I/R-induced barrier dysfunction are also discussed. It concludes with an analysis of how risk factors for cardiovascular disease, i.e. hypertension, diabetes, hypercholesterolaemia, and obesity, influence the vascular permeability response to I/R. Areas of uncertainty and controversy in this field of investigation are also identified.

The nitric oxide (NO) cascade and endothelial NO synthase (eNOS) are best known for their role in endothelium-mediated relaxation of vascular smooth muscle. Activation of eNOS by certain inflammatory stimuli and enhanced NO release have also been shown to promote increased microvascular permeability. However, it is not entirely clear why activation of eNOS by certain vasodilatory agents, like acetylcholine, does not affect microvascular permeability, whereas activation of eNOS by other inflammatory agents that increase permeability, like platelet-activating factor, does not cause vasodilation. In this review, we discuss the evidence demonstrating the role of eNOS in the elevation of microvascular permeability. We also examine the relative importance of eNOS phosphorylation and localization in its function to promote elevated microvascular permeability as well as emerging topics with regard to eNOS and microvascular permeability regulation.

Inflammatory mediators increase vascular permeability primarily by formation of intercellular gaps between endothelial cells of post-capillary venules. Under these conditions, endothelial cell-cell contacts such as adherens and tight junctions open to allow paracellular fluid passage. Small guanosine triphosphatases (GTPases) from the ras superfamily, primarily Rho GTPases (RhoA, Rac1, Cdc42) or Rap1 are known to regulate cell adhesion, in part by reorganization of the junction-associated cortical actin cytoskeleton. In this review, we will discuss the role of small GTPases for the maintenance of microvascular barrier functions under resting conditions as well as under conditions of increased permeability and their involvement in signalling pathways downstream of both barrier-stabilizing and inflammatory mediators. Rac1 and Cdc42 are the main GTPases required for barrier maintenance and stabilization, whereas RhoA negatively regulates barrier properties under both resting and inflammatory conditions. For Rac1 and RhoA, contrary functions under certain conditions have also been described. However, Rac1-mediated barrier destabilization in microvascular endothelium appears to be largely restricted to conditions of enhanced endothelial cell migration and thus to be more closely related to angiogenesis rather than to inflammation. Recent studies revealed that cAMP signalling, which is well known to be barrier protective, enhances barrier functions in part via Rap1-mediated activation of Rac1 and Cdc42 as well as by inhibition of RhoA. Moreover, barrier-stabilizing mediators directly activate Rac1 and Cdc42 or increase cAMP levels. On the other hand, several barrier-disruptive components appear to increase permeability by reduced formation of cAMP, leading to both inactivation of Rac1 and activation of RhoA.