Sarah Y. Yuan

Myosin Light Chain Kinase in Microvascular Endothelial Barrier Function

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.

Rho and ROCK Signaling in VEGF-Induced Microvascular Endothelial Hyperpermeability

Objectives: Vascular endothelial growth factor (VEGF) plays an important role in the regulation of microvascular permeability under various physiological and pathological conditions. The authors tested the hypothesis that the small GTPase Rho and its downstream effector ROCK (Rho‐associated coiled‐coil‐containing protein kinase) mediate VEGF‐induced increases in venular permeability. They also investigated myosin light chain (MLC) phosphorylation and actin polymerization, two well‐characterized targets of the Rho‐ROCK pathway that are implicated in the regulation of endothelial barrier function.

Nonmuscle Myosin Light-Chain Kinase Deficiency Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice via Reduced Endothelial Barrier Dysfunction and Monocyte Migration

Background— Endothelial dysfunction and monocyte migration are key events in the pathogenesis of atherosclerosis. Nonmuscle myosin light-chain kinase (nmMLCK), the predominant MLCK isoform in endothelial cells, has been shown to contribute to vascular inflammation by altering endothelial barrier function. However, its impact on atherogenesis remains unknown. Methods and Results— We investigated the role of nmMLCK in the development of atherosclerotic lesions in apolipoprotein E–deficient (apoE−/−) mice fed an atherogenic diet for 12 weeks. Histopathological examination demonstrated that nmMLCK deficiency (apoE−/−nmmlck−/−) reduced the size of aortic lesions by 53%, lipid contents by 44%, and macrophage deposition by 40%. Western blotting and reverse-transcription polymerase chain reaction revealed the expression of nmMLCK in aortic endothelial cells and peripheral blood monocytes. Measurements of transendothelial electric resistance indicated that nmMLCK deficiency attenuated endothelial barrier dysfunction caused by thrombin, oxidized low-density lipoprotein, and tumor necrosis factor &agr;. In monocytes, nmMLCK deficiency reduced their migration in response to the chemokine monocyte chemoattractant protein-1. Further mechanistic studies showed that nmMLCK acted through both myosin light chain phosphorylation-coupled and -uncoupled pathways; the latter involved Rous sacracoma virus homolog genes-encoded tyrosine kinases (Src) signaling. Moreover, depletion of Src via gene silencing, site-specific mutagenesis, or pharmacological inhibition of Src greatly attenuated nmMLCK-dependent endothelial barrier dysfunction and monocyte migration. Conclusions— Nonmuscle myosin light-chain kinase contributes to atherosclerosis by regulating endothelial barrier function and monocyte migration via mechanisms involving not only kinase-mediated MLC phosphorylation but also Src activation.

Microvascular Permeability in Diabetes and Insulin Resistance

ABSTRACT

ADAM15 regulates endothelial permeability and neutrophil migration via Src/ERK1/2 signalling

AIMS Endothelial barrier dysfunction is a key event in the pathogenesis of vascular diseases associated with inflammation. ADAM (a disintegrin and metalloprotease) 15 has been shown to contribute to the development of vascular inflammation. However, its role in regulating endothelial barrier function is unknown. The aim of this study was to examine the effect of ADAM15 on endothelial permeability and its underlying mechanisms. METHODS AND RESULTS By measuring albumin transendothelial flux and transendothelial electric resistance in cultured human umbilical vein endothelial cell monolayers, we found that depletion of ADAM15 expression via siRNA decreased endothelial permeability and attenuated thrombin-induced barrier dysfunction. In contrast, endothelial cells overexpressing either wild-type or catalytically dead mutant ADAM15 displayed a higher basal permeability and augmented hyperpermeability in response to thrombin. In addition, ADAM15 knockdown inhibited whereas ADAM15 overexpression promoted neutrophil transendothelial migration. Further molecular assays revealed that ADAM15 did not cleave vascular endothelial-cadherin or cause its degradation. However, overexpression of ADAM15 promoted extracellular signal-regulated kinase (ERK)1/2 phosphorylation in both non-stimulated and thrombin-stimulated endothelial cells in a protease activity-independent manner. Pharmacological inhibition of Src kinase or ERK activation reversed ADAM15-induced hyperpermeability and neutrophil transmigration. CONCLUSION 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.

Neutrophil transmigration, focal adhesion kinase and endothelial barrier function.

Neutrophil activation is an essential component of innate immune defense against infection and injury. In response to inflammatory stimulation, circulating neutrophils undergo a series of dynamic and metabolic changes characterized by β2-intergrin mediated adhesion to microvascular endothelium and subsequent transendothelial migration. During this process, neutrophils release granular contents containing digestive enzymes and produce cytotoxic agents such as reactive oxygen species and cytokines. These products target endothelial barriers inducing phosphorylation-triggered junction dissociation, actin stress fiber formation, and actomyosin contraction, manifest as paracellular hyperpermeability. Endothelial cell-matrix focal adhesions play an integral role in this process by providing structural support for endothelial conformational changes that facilitate neutrophil transmigration, as well as by recruiting intracellular molecules that constitute the hyperpermeability signaling cascades. As a central connector of the complex signaling network, focal adhesion kinase (FAK) is activated following neutrophil adhesion, and further mediates the reorganization of endothelial integrin-matrix attachments in a pattern coordinating with cytoskeleton contraction and junction opening. In this review, we present recent experimental evidence supporting the importance of FAK in neutrophil-dependent regulation of endothelial permeability. The discussion focuses on the mechanisms by which neutrophils activate FAK and its downstream effects on endothelial barriers.

A role for endothelial-derived matrix metalloproteinase-2 in breast cancer cell transmigration across the endothelial-basement membrane barrier

Invasive cancer cells utilize matrix metalloproteinases (MMPs) to degrade the extracellular matrix and basement membrane in the process of metastasis. Among multiple members of the MMP family, the gelatinase MMP-2 has been implicated in the development and dissemination of malignancies. However, the cellular source of MMP-2 and its effect on metastatic extravasation have not been well characterized. The objective of this study was to test the hypothesis that active MMP-2 derived from endothelial cells facilitated the transmigration of breast cancer cells across the microvascular barrier. Gelatin zymography was used to assess latent and active MMP-2 production in conditioned media from MDA-MB-231 human breast cancer cells, human lung microvascular endothelial cells (HLMVEC) and co-culture of these two cells. Transmigrated cancer cells were measured during MMP-2 knockdown with siRNA and pharmacological inhibition of MMP activity with OA-HY. The results showed consistent MMP-2 secretion by the HLMVECs, whereas a low level production was seen in the MDA-MB-231 cells. Inhibition of MMP-2 expression or activity in HLMVECs significantly attenuated the transmigration of MDA-MB-231 cells across an endothelial monolayer barrier grown on a reconstituted basement membrane. The data provide evidence supporting a potential role for the endothelial production of MMPs in promoting cancer cell extravasation. We suggest that the interaction between malignant cells and peritumoral benign tissues including the vascular endothelium may serve as an important mechanism in the regulation of tumor invasion and metastasis.

Tissue Inhibitor of Metalloproteinase-2 Regulates Matrix Metalloproteinase-2–Mediated Endothelial Barrier Dysfunction and Breast Cancer Cell Transmigration through Lung Microvascular Endothelial Cells

Matrix metalloproteinases (MMP) have been implicated in multiple stages of cancer metastasis. Tissue inhibitor of metalloproteinase-2 (TIMP-2) plays an important role in regulating MMP-2 activity. By forming a ternary complex with pro-MMP-2 and its activator MMP-14 on the cell surface, TIMP-2 can either initiate or restrain the cleavage and subsequent activation of MMP-2. Our recent work has shown that breast cancer cell adhesion to vascular endothelial cells activates endothelial MMP-2, promoting tumor cell transendothelial migration (TEME). However, the mechanism of MMP-2 regulation during TEME remains unclear. In the current study, we present evidence that MMP-14 is expressed in both invasive breast cancer cells (MDA-MB-231 and MDA-MB-436) and lung microvascular endothelial cells (HBMVEC-L), whereas TIMP-2 is exclusively expressed and released from the cancer cells. The tumor cell–derived TIMP-2 was further identified as a major determinant of endothelial MMP-2 activity during tumor cell transmigration in the presence of MMP-14. This response was associated with endothelial barrier dysfunction because coculture of MDA-MB-231 or MDA-MB-436 with HBMVEC-L caused a significant decrease in transendothelial electrical resistance concomitantly with endothelial cell-cell junction disruption and tumor cell transmigration. Knockdown of TIMP-2 or inhibition of TIMP-2/MMP-14 attenuated MMP-2–dependent transendothelial electrical resistance response and TEME. These findings suggest a novel interactive role of breast cancer cells and vascular endothelial cells in regulating the TIMP-2/MMP-14/MMP-2 pathway during tumor metastasis. Mol Cancer Res; 8(7); 939–51. ©2010 AACR.

Serum metalloproteinases MMP-2, MMP-9, and metalloproteinase tissue inhibitors in patients are associated with arteriovenous fistula maturation

OBJECTIVE Many vascular surgeons construct arteriovenous fistulas (AVFs) for hemodialysis access as the primary choice access. A significant number of AVFs fail to mature, however, leading to patient frustration and repeated operations. Metalloproteinase (MMP) activity, particularly MMP-2 and MMP-9, may be important for AVF maturation. We therefore sought to identify whether serum MMP levels could serve as a biomarker for predicting future successful AVF maturation. METHODS Blood was collected from patients with chronic renal insufficiency at the time of surgery for long-term hemodialysis access. Serum was separated from whole blood and ultracentrifuged at 1000g for 10 minutes. Serum aliquots were frozen at -80°C until used for analysis. Enzyme-linked immunosorbent assay was used to assay levels of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase type 2 (TIMP-2), and TIMP type 4 (TIMP-4). Clinical end points were used to divide patients into failed and matured AVF groups. Successful maturation was considered in patients who had specific duplex findings or 1 month of successful two-needle cannulation hemodialysis. MMP/TIMP ratios were calculated as an index of the MMP axis activity because MMP activity parallels alterations in TIMP levels. RESULTS Of 20 enrolled patients, AVF maturation was successful in 13 and failed in 7. Serum levels of MMP-2/TIMP-2 were significantly higher in patients with matured AVFs vs levels in those that failed (P = .003). Similarly, a trend toward increased serum levels of MMP-9/TIMP-4 was found in patients with successful AVF (P = .06). CONCLUSIONS MMP-2 and TIMP-2 levels were different among patients whose AVF matured vs those who did not. Further follow-up studies to determine the predictability of AVF maturation using relative patient serum levels of MMP-2 and TIMP-2 should be performed.

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.