Rachel M. Perrin

Microvascular Permeability in Diabetes and Insulin Resistance

ABSTRACT

A role for long chain myosin light chain kinase (MLCK-210) in microvascular hyperpermeability during severe burns.

Microvascular leakage has been implicated in the pathogenesis of multiple organ dysfunction during trauma. Previous studies suggest the involvement of myosin light chain (MLC) phosphorylation-triggered endothelial contraction in the development of microvascular hyperpermeability. Myosin light chain kinase (MLCK) plays a key role in the control of MLC-phosphorylation status; thus, it is thought to modulate barrier function through its regulation of intracellular contractile machinery. The aim of this study was to further investigate the endothelial mechanism of MLC-dependent barrier injury in burns, focusing on the long isoform of MLCK (MLCK-210) that has recently been identified as the predominant isoform expressed in vascular endothelial cells. An MLCK-210 knockout mouse model was subjected to third-degree scald burn covering 25% total body surface area. The mesenteric microcirculation was observed using intravital microscopy, and the microvascular permeability was assessed by measuring the transvenular flux of fluorescein isothiocyanate-albumin. In a separate experiment, in vivo mesenteric hydraulic conductivity (Lp) was measured using the modified Landis technique. The injury caused a profound microvascular leakage, as indicated by a 2-fold increase in albumin flux and 4-fold increase in Lp at the early stages, which was associated with a high mortality within the 24-h period. Compared with wild-type control, the MLCK-210-deficient mice displayed a significantly improved survival with a greatly attenuated microvascular hyperpermeability response to albumin and fluid. These results provide direct evidence for a role of MLCK-210 in mediating burn-induced microvascular barrier injury and validate MLCK-210 as a potential therapeutic target in the treatment of burn edema.

A Role for the Endothelial Glycocalyx in Regulating Microvascular Permeability in Diabetes Mellitus

Diabetic angiopathy is a major cause of morbidity and mortality in diabetes mellitus. Endothelial dysfunction and associated alterations in blood flow, pressure and permeability are widely accepted phenomena in the diabetic milieu and are understood to lead to microangiopathy. Despite the clinical importance of diabetic microangiopathy, the mechanisms of pathogenesis remain elusive. In particular, much is yet to be understood about the nature of the putative increased permeability with respect to diabetes. Microvessel permeability is intrinsically difficult to measure and a surrogate (solute or solvent flux) is usually reported, the measurement of which is hampered by haemodynamic factors, such as flow rate, hydrostatic pressure gradient, solute concentration and surface area available for exchange. Very few studies describing the measurement of permeability with respect to diabetes have controlled for all these factors. As a result, the nature of the increased microvessel permeability in diabetes mellitus and indeed its causes are poorly understood. Recent studies have shown that hyperglycaemia can alter the glycocalyx structure, and parallel findings have shown that the apparent increase in permeability demonstrated in hyperglycaemia may be due to an increase in the permeability of the vessels to water, and not an increase in protein permeability, an effect attributable to altered glycocalyx. This review focuses on the current understanding of microvascular permeability in terms of the endothelial glycocalyx- fibre-matrix theory, those methods used to determine permeability in the context of diabetes, and the more recent developments in our understanding of elevated microvascular permeability in the diabetic circulation.

Fibrinogen-γ C-Terminal Fragments Induce Endothelial Barrier Dysfunction and Microvascular Leak via Integrin-Mediated and RhoA-Dependent Mechanism

Objectives—The purposes of this study were to characterize the direct effect of the C-terminal fragment of fibrinogen &ggr; chain (&ggr;C) on microvascular endothelial permeability and to examine its molecular mechanism of action. Methods and Results—Intravital microscopy was performed to measure albumin extravasation in intact mesenteric microvasculature, followed by quantification of hydraulic conductivity in single perfused microvessels. Transendothelial electric resistance was measured in microvascular endothelial cells in combination with immunoblotting and immunocytochemistry. The results show that &ggr;C induced time- and concentration-dependent increases in protein transvascular flux and water permeability and decreases in endothelial barrier function, coupled with Rho GTPase activation, myosin light chain phosphorylation, and stress fiber formation. Depletion of RhoA via siRNA knockdown or pharmacological inhibition of RhoA signaling attenuated &ggr;C-induced barrier dysfunction. Imaging analyses demonstrated binding of &ggr;C to endothelial cells; the interaction was inhibited during blockage of the αvβ3 integrin. Furthermore, in vivo experiments showed that the microvascular leak response to &ggr;C was attenuated in integrin β3−/− animals. Conclusion—Fibrinogen-&ggr; C terminus directly interacts with the microvascular endothelium causing fluid and protein leak. The endothelial response to &ggr;C involves an integrin receptor-mediated RhoA-dependent signaling pathway that leads to paracellular hyperpermeability.

Counter Regulatory Effects of PKCβII and PKCδ on Coronary Endothelial Permeability

Objective—The aim of this study was to examine the endothelial distribution and activity of selected PKC isoforms in coronary vessels with respect to their functional impact on endothelial permeability under the experimental conditions relevant to diabetes. Methods and Results—En face immunohistochemistry demonstrated a significant increase of PKCβ&Igr;&Igr; and decrease of PKCΔ expression in coronary arterial endothelium of Zucker diabetic rats. To test whether changes in PKC expression alter endothelial barrier properties, we measured the transcellular electric resistance in human coronary microvascular endothelial monolayers and found that either PKCβ&Igr;&Igr; overexpression or PKCΔ inhibition disrupted the cell–cell adhesive barrier. Three-dimensional fluorescence microscopy revealed that hyperpermeability was caused by altered PKC activity in association with distinct translocation of PKCβ&Igr;&Igr; to the cell–cell junction and PKCΔ localization to the cytosol. Further analyses in fractionated endothelial lysates confirmed the differential redistribution of these isozymes. Additionally, FRET analysis of PKC subcellular dynamics demonstrated a high PKCβ&Igr;&Igr; activity at the cell surface and junction, whereas PKCΔ activity is concentrated in intracellular membrane organelles. Conclusion—Taken together, these data suggest that PKCβ&Igr;&Igr; and PKCΔ counter-regulate coronary endothelial barrier properties by targeting distinctive subcellular sites. Imbalanced PKCβ&Igr;&Igr;/PKCΔ expression and activity may contribute to endothelial hyperpermeability and coronary dysfunction in diabetes.

Counter regulatory effects of PKCbetaII and PKCdelta on coronary endothelial permeability.

Objective—The aim of this study was to examine the endothelial distribution and activity of selected PKC isoforms in coronary vessels with respect to their functional impact on endothelial permeability under the experimental conditions relevant to diabetes. Methods and Results—En face immunohistochemistry demonstrated a significant increase of PKCβ&Igr;&Igr; and decrease of PKCΔ expression in coronary arterial endothelium of Zucker diabetic rats. To test whether changes in PKC expression alter endothelial barrier properties, we measured the transcellular electric resistance in human coronary microvascular endothelial monolayers and found that either PKCβ&Igr;&Igr; overexpression or PKCΔ inhibition disrupted the cell–cell adhesive barrier. Three-dimensional fluorescence microscopy revealed that hyperpermeability was caused by altered PKC activity in association with distinct translocation of PKCβ&Igr;&Igr; to the cell–cell junction and PKCΔ localization to the cytosol. Further analyses in fractionated endothelial lysates confirmed the differential redistribution of these isozymes. Additionally, FRET analysis of PKC subcellular dynamics demonstrated a high PKCβ&Igr;&Igr; activity at the cell surface and junction, whereas PKCΔ activity is concentrated in intracellular membrane organelles. Conclusion—Taken together, these data suggest that PKCβ&Igr;&Igr; and PKCΔ counter-regulate coronary endothelial barrier properties by targeting distinctive subcellular sites. Imbalanced PKCβ&Igr;&Igr;/PKCΔ expression and activity may contribute to endothelial hyperpermeability and coronary dysfunction in diabetes.

Counter Regulatory Effects of PKC βII and PKC δ on Coronary Endothelial Permeability

Objective—The aim of this study was to examine the endothelial distribution and activity of selected PKC isoforms in coronary vessels with respect to their functional impact on endothelial permeability under the experimental conditions relevant to diabetes. Methods and Results—En face immunohistochemistry demonstrated a significant increase of PKCβ&Igr;&Igr; and decrease of PKCΔ expression in coronary arterial endothelium of Zucker diabetic rats. To test whether changes in PKC expression alter endothelial barrier properties, we measured the transcellular electric resistance in human coronary microvascular endothelial monolayers and found that either PKCβ&Igr;&Igr; overexpression or PKCΔ inhibition disrupted the cell–cell adhesive barrier. Three-dimensional fluorescence microscopy revealed that hyperpermeability was caused by altered PKC activity in association with distinct translocation of PKCβ&Igr;&Igr; to the cell–cell junction and PKCΔ localization to the cytosol. Further analyses in fractionated endothelial lysates confirmed the differential redistribution of these isozymes. Additionally, FRET analysis of PKC subcellular dynamics demonstrated a high PKCβ&Igr;&Igr; activity at the cell surface and junction, whereas PKCΔ activity is concentrated in intracellular membrane organelles. Conclusion—Taken together, these data suggest that PKCβ&Igr;&Igr; and PKCΔ counter-regulate coronary endothelial barrier properties by targeting distinctive subcellular sites. Imbalanced PKCβ&Igr;&Igr;/PKCΔ expression and activity may contribute to endothelial hyperpermeability and coronary dysfunction in diabetes.

Counter Regulatory Effects of PKCβIIand PKCδon Coronary Endothelial Permeability

Objective—The aim of this study was to examine the endothelial distribution and activity of selected PKC isoforms in coronary vessels with respect to their functional impact on endothelial permeability under the experimental conditions relevant to diabetes. Methods and Results—En face immunohistochemistry demonstrated a significant increase of PKCβ&Igr;&Igr; and decrease of PKCΔ expression in coronary arterial endothelium of Zucker diabetic rats. To test whether changes in PKC expression alter endothelial barrier properties, we measured the transcellular electric resistance in human coronary microvascular endothelial monolayers and found that either PKCβ&Igr;&Igr; overexpression or PKCΔ inhibition disrupted the cell–cell adhesive barrier. Three-dimensional fluorescence microscopy revealed that hyperpermeability was caused by altered PKC activity in association with distinct translocation of PKCβ&Igr;&Igr; to the cell–cell junction and PKCΔ localization to the cytosol. Further analyses in fractionated endothelial lysates confirmed the differential redistribution of these isozymes. Additionally, FRET analysis of PKC subcellular dynamics demonstrated a high PKCβ&Igr;&Igr; activity at the cell surface and junction, whereas PKCΔ activity is concentrated in intracellular membrane organelles. Conclusion—Taken together, these data suggest that PKCβ&Igr;&Igr; and PKCΔ counter-regulate coronary endothelial barrier properties by targeting distinctive subcellular sites. Imbalanced PKCβ&Igr;&Igr;/PKCΔ expression and activity may contribute to endothelial hyperpermeability and coronary dysfunction in diabetes.