Endothelial hyperpermeability is a significant problem in vascular inflammation associated with trauma, ischaemia-reperfusion injury, sepsis, adult respiratory distress syndrome, diabetes, thrombosis and cancer. An important mechanism underlying this process is increased paracellular leakage of plasma fluid and protein. Inflammatory stimuli such as histamine, thrombin, vascular endothelial growth factor and activated neutrophils can cause dissociation of cell-cell junctions between endothelial cells as well as cytoskeleton contraction, leading to a widened intercellular space that facilitates transendothelial flux. Such structural changes initiate with agonist-receptor binding, followed by activation of intracellular signalling molecules including calcium, protein kinase C, tyrosine kinases, myosin light chain kinase, and small Rho-GTPases; these kinases and GTPases then phosphorylate or alter the conformation of different subcellular components that control cell-cell adhesion, resulting in paracellular hypermeability. Targeting key signalling molecules that mediate endothelial-junction-cytoskeleton dissociation demonstrates a therapeutic potential to improve vascular barrier function during inflammatory injury.Endothelial cells lining the inner surface of microvessels form a semipermeable barrier that actively participates in blood-tissue exchange of plasma fluid, proteins and cells. The precise regulation of endothelial permeability is essential for maintaining circulatory homeostasis and the physiological function of different organs. As a result, microvascular barrier dysfunction and endothelial hyperpermeability represent crucial events in the development of a variety of disease processes, such as adult respiratory distress syndrome (ARDS), ischemia-reperfusion (I-R) injury, diabetic vascular complications, and tumour metastasis. Better insight into the molecular mechanisms underlying pathogenic conditions related to microvascular hyperpermeability is required for developing effective therapeutic strategies. Following intensive studies over the past few decades, it is now understood that endothelial permeability is mediated through a transcellular pathway (across cells) and a paracellular pathway (between cells), both of which are highly regulated by mechanical forces and biochemical signals. Transcellular versus paracellular permeabilityAn important molecular mechanism underlying transcellular permeability is macromolecule transcytosis via caveoli -specialised plasmalemmal vesicles containing caveolin-1. The involvement of caveolin-1 in regulating cardiovascular functions associated with endothelial barrier properties has been demonstrated through studies using transgenic and knockout NIH Public Access Author ManuscriptExpert Rev Mol Med. Author manuscript; available in PMC 2010 February 24. Published in final edited form as:Expert Rev Mol Med. ; 11: e19. doi:10.1017/S1462399409001112. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript animals (Refs 1,2,3,4). Upon binding to...

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.

Notch activation, which is associated with basal-like breast cancer (BLBC), normally directs tissue patterning, suggesting that it may shape the tumor microenvironment. Here, we show that Notch in tumor cells regulates the expression of two powerful proinflammatory cytokines, IL1β and CCL2, and the recruitment of tumor-associated macrophages (TAM). Notch also regulates TGFβ-mediated activation of tumor cells by TAMs, closing a Notch-dependent paracrine signaling loop between these two cell types. We use a mouse model in which Notch can be regulated in spontaneous mammary carcinoma to confirm that IL1β and CCL2 production, and macrophage recruitment are Notch-dependent. In human disease, expression array analyses demonstrate a striking association between Notch activation, IL1β and CCL2 production, macrophage infiltration, and BLBC. These findings place Notch at the nexus of a vicious cycle of macrophage infiltration and amplified cytokine secretion and provide immunotherapeutic opportunities in BLBC. BLBC is aggressive and has an unmet need for effective targeted treatment. Our data highlight immunotherapeutic opportunities in Notch-activated BLBC. Effective IL1β and CCL2 antagonists are currently in clinical review to treat benign inflammatory disease, and their transition to the cancer clinic could have a rapid impact. .

Microvascular barrier dysfunction is a serious problem that occurs in many inflammatory conditions, including sepsis, trauma, ischemia–reperfusion injury, cardiovascular disease, and diabetes. Barrier dysfunction permits extravasation of serum components into the surrounding tissue, leading to edema formation and organ failure. The basis for microvascular barrier dysfunction is hyperpermeability at endothelial cell–cell junctions. Endothelial hyperpermeability is increased by actomyosin contractile activity in response to phosphorylation of myosin light chain by myosin light chain kinase (MLCK). MLCK-dependent endothelial hyperpermeability occurs in response to inflammatory mediators (e.g., activated neutrophils, thrombin, histamine, tumor necrosis factor alpha, etc.), through multiple cell signaling pathways and signaling molecules (e.g., Ca++, protein kinase C, Src kinase, nitric oxide synthase, etc.). Other signaling molecules protect against MLCK-dependent hyperpermeability (e.g., sphingosine-1-phosphate or cAMP). In addition, individual MLCK isoforms play specific roles in endothelial barrier dysfunction, suggesting that isoform-specific inhibitors could be useful for treating inflammatory disorders and preventing multiple organ failure. Because endothelial barrier dysfunction depends upon signaling through MLCK in many instances, MLCK-dependent signaling comprises multiple potential therapeutic targets for preventing edema formation and multiple organ failure. The following review is a discussion of MLCK-dependent mechanisms and cell signaling events that mediate endothelial hyperpermeability.

The MUC1 mucin is normally restricted to the apical surface of breast epithelial cells. In tumors, it is frequently overexpressed and underglycosylated. The MUC1 peptide core mediates firm adhesion of tumor cells to adjacent cells via binding to intercellular adhesion molecule-1 (ICAM-1). There is increasing evidence that MUC1 is involved in signaling, with current reports focusing on phosphorylation of the MUC1 cytoplasmic tail after indirect or artificial modes of stimulation. ICAM-1 is the only known direct ligand of the MUC1 extracellular domain. The data presented herein show that MUC1 expressed on the surface of breast cancer cell lines or transfected 293T cells can initiate a calcium-based oscillatory signal on contact with ICAM-1-transfected NIH 3T3 cells, and we present a novel method of quantifying and comparing calcium oscillations. The MUC1-induced signal appears to be distinct from those previously described, and may involve a Src family kinase, phosphoinositol 3-kinase, phospholipase C, and lipid rafts, but not mitogenactivated protein kinase. As calcium signaling has been associated with cytoskeletal change and motility, it is possible that the functions of MUC1 include heterotypic cell-cell adhesion followed by a calcium-based promigratory signal within tumor cells, thus facilitating metastasis.The MUC1 mucin has been well established as a tumor marker in breast cancer and is implicated in metastatic spread (1). Phosphorylation of the MUC1 cytoplasmic tail (2-4) allows MUC1 to associate with potential oncogenes (5-8). MUC1 can also be indirectly stimulated to initiate signaling cascades, through epidermal growth factor receptor (EGFR) 1 (9, 10) or antibodies directed against CD8-MUC1 chimeras (11, 12), resulting in activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) pathway. Our laboratory has previously identified intercellular adhesion molecule-1 (ICAM-1) as a natural, endogenous ligand for MUC1 (13,14). ICAM-1 is currently the only reported direct ligand for the MUC1 extracellular domain, and binding promotes adhesion of tumor cells to a simulated vessel wall construct under fluid flow conditions (15). In this report, we demonstrate a novel signaling paradigm, in which MUC1 participates in initiating intracellular calcium oscillations after direct stimulation by ICAM-1. As calcium signaling has previously been implicated in cytoskeletal remodeling and motility (16), we hypothesize that this signal is involved in tumor cell migration. Thus, the MUC1/ICAM-1 interaction may facilitate extravasation of tumor cells after mediating binding to the blood vessel wall. EXPERIMENTAL PROCEDURESReagents-The CT2 antibody against the MUC1 cytoplasmic domain and the pC1Neo TRϩ FLAG plasmid carrying the MUC1 gene were generously provided by Dr. Sandra Gendler, Mayo Clinic, Scottsdale, AZ. The MUC1 gene was PCR amplified from the plasmid and inserted into the Clontech pEYFP-N1 plasmid at the BsrGI/NotI cut sites. A synthetic MUC1-specific signal sequence, TCGACTAG...

MUC1, a transmembrane glycoprotein of the mucin family, when aberrantly expressed on breast cancer cells is correlated with increased lymph node metastases. We have previously shown that MUC1 binds intercellular adhesion molecule-1 (ICAM-1) on surrounding accessory cells and facilitates transendothelial migration of MUC1-bearing cells. Nevertheless, the underlying molecular mechanism is still obscure. In the present study, we used a novel assay of actin cytoskeletal reorganization to show that by ligating ICAM-1, MUC1 triggers Rac1-and Cdc42-dependent actin cytoskeletal protrusive activity preferentially at the heterotypic cell-cell contact sites. Further, we show that these MUC1/ICAM-1 interaction -initiated lamellipodial and filopodial protrusions require Src family kinase and CT10 regulator of kinase like (CrkL) accompanied by the rapid formation of a Src-CrkL signaling complex at the MUC1 cytoplasmic domain. Through inhibition of Src kinase activity, we further revealed that Src is required for recruiting CrkL to the MUC1 cytoplasmic domain as well as mediating the observed actin cytoskeleton dynamics. These findings suggest a novel MUC1-Src-CrkL-Rac1/Cdc42 signaling cascade following ICAM-1 ligation, through which MUC1 regulates cytoskeletal reorganization and directed cell motility during cell migration. (Mol Cancer Res 2008;6(4):555 -67)

Cancer cachexia is a kind of whole-body metabolic disorder syndrome accompanied by severe wasting of muscle tissue in which cancer exosomes may be involved. Analysis of clinical samples showed that the serum exosome concentrations were correlated with the development of cancer cachexia. Exosomes secreted by C26 cells could decrease the diameter of C2C12 myotubes in vitro and decrease mouse muscle strength and tibialis anterior (TA) muscle weight in vivo . GW4869, an inhibitor of exosome excretion, ameliorated muscle wasting in C26 tumor-bearing mice. MicroRNA (miRNA) sequencing (miRNA-seq) analysis suggested that miR-195a-5p and miR-125b-1-3p were richer in C26 exosomes than in exosomes secreted from MC38 cells (non-cachexic). Both miR-195a-5p and miR-125b-1-3p mimics could induce atrophy of C2C12 myoblasts. Downregulation of Bcl-2 and activation of the apoptotic signaling pathway were observed in C2C12 myoblasts transfected with miR-195a-5p and miR-125b-1-3p mimics, in the gastrocnemius muscle of C26 tumor-bearing mice and in the TA muscle injected with C26 exosomes. Results of dual-luciferase assay confirmed the targeting of miR-195a-5p/miR-125b-1-3p to Bcl-2. Overexpression of Bcl-2 successfully reversed atrophy of C2C12 myoblasts induced by the two miRNA mimics. These results suggested that cancer exosome enriched miRNAs might induce muscle atrophy by targeting Bcl-2-mediated apoptosis.

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.