Abstract-Neutrophil-induced coronary microvascular leakage represents an important pathophysiological consequence of ischemic and inflammatory heart diseases. The precise mechanism by which neutrophils regulate endothelial barrier function remains to be established. The aim of this study was to examine the microvascular endothelial response to neutrophil activation with a focus on myosin light chain kinase (MLCK)-mediated myosin light chain (MLC) phosphorylation, a regulatory process that controls cell contraction. The apparent permeability coefficient of albumin (Pa) was measured in intact isolated porcine coronary venules. Incubation of the vessels with C5a-activated neutrophils induced a time-and concentration-dependent increase in Pa. The hyperpermeability response was significantly attenuated during inhibition of endothelial MLC phosphorylation with the selective MLCK inhibitor ML-7 and transfection of a specific MLCK-inhibiting peptide. In contrast, transfection of constitutively active MLCK elevated Pa, which was abolished by ML-7. In addition to the vessel study, albumin transendothelial flux was measured in cultured bovine coronary venular endothelial monolayers, which displayed a hyperpermeability response to neutrophils and MLCK in a pattern similar to that in venules. Importantly, neutrophil stimulation caused MLC phosphorylation in endothelial cells in a time course closely correlated with that of the hyperpermeability response. Consistently, the MLCK inhibitors abolished neutrophil-induced MLC phosphorylation. Furthermore, immunohistochemical observation of neutrophil-stimulated endothelial cells revealed an increased staining for phosphorylated MLC in association with contractile stress fiber formation and intercellular gap development. Taken together, the results suggest that endothelial MLCK activation and MLC phosphorylation play an important role in mediating endothelial barrier dysfunction during neutrophil activation.

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Microvascular barrier injury has been implicated in the initiation and progress of end organ complications of diabetic mellitus. Plasma leakage and fluid retention are seen in various tissues of diabetic patients or animals at the early stages of the disease before structural microangiopathy can be detected. Clinical and experimental evidence suggests that hyperglycemia, often accompanied with insulin deficiency or insulin resistance, causes impaired autoregulation and increased permeability in microvessels. Multiple molecular pathways have been identified as contributors to the altered fluid homeostasis, including increased polyol flux that promotes oxidative stress, advanced glycation that leads to carbonyl stress, and excessive glucose metabolism that results in protein kinase C activation. These abnormal metabolic activities are associated with the production of pro-inflammatory cytokines and growth factors, which can stimulate an array of signaling reactions and structural changes at the endothelial barrier and ultimately cause microvascular leakage. Interventions that manipulate these metabolic and inflammatory pathways have demonstrated efficacy in delaying the progress of diabetic microvascular complications; however, their direct effects and mechanisms of action on the microcirculation remain elusive. A deeper understanding of the molecular basis of diabetes-induced endothelial barrier dysfunction will provide a framework for the development of new therapeutic targets to treat this chronic and debilitating disease process.

Atomic force microscopy was used to investigate the cellular response to histamine, one of the major inflammatory mediators that cause endothelial hyperpermeability and vascular leakage. AFM probes were labeled with fibronectin and used to measure binding strength between alpha5beta1 integrin and fibronectin by quantifying the force required to break single fibronectin-integrin bonds. The cytoskeletal changes, binding probability, and adhesion force before and after histamine treatment on endothelial cells were monitored. Cell topography measurements indicated that histamine induces cell shrinkage. Local cell stiffness and binding probability increased twofold after histamine treatment. The force necessary to rupture single alpha5beta1-fibronectin bond increased from 34.0 +/- 0.5 pN in control cells to 39 +/- 1 pN after histamine treatment. Experiments were also conducted to confirm the specificity of the alpha5beta1-fibronectin interaction. In the presence of soluble GRGDdSP the probability of adhesion events decreased >50% whereas the adhesion force between alpha5beta1 and fibronectin remained unchanged. These data indicate that extracellular matrix-integrin interactions play an important role in the endothelial cell response to changes of external chemical mediators. These changes can be recorded as direct measurements on live endothelial cells by using atomic force microscopy.

The data provide evidence for a novel function of ADAM15 in regulating endothelial barrier properties. The mechanisms of ADAM15-induced hyperpermeability involve Src/ERK1/2 signalling independent of junction molecule shedding.