VE-cadherin and claudin-5: it takes two to tango

Endothelial barrier function requires the adhesive activity of VE-cadherin and claudin-5, which are key components of adherens and tight endothelial junctions, respectively. Emerging evidence suggests that VE-cadherin controls claudin-5 expression by preventing the nuclear accumulation of FoxO1 and -catenin, which repress the claudin-5 promoter. This indicates that a crosstalk mechanism operates between these junctional structures

Mtss1 promotes cell-cell junction assembly and stability through the small GTPase Rac1

Cell-cell junctions are an integral part of epithelia and are often disrupted in cancer cells during epithelial-to-mesenchymal transition (EMT), which is a main driver of metastatic spread. We show here that Metastasis suppressor-1 (Mtss1; Missing in Metastasis, MIM), a member of the IMD-family of proteins, inhibits cell-cell junction disassembly in wound healing or HGF-induced scatter assays by enhancing cell-cell junction strength. Mtss1 not only makes cells more resistant to cell-cell junction disassembly, but also accelerates the kinetics of adherens junction assembly. Mtss1 drives enhanced junction formation specifically by elevating Rac-GTP. Lastly, we show that Mtss1 depletion reduces recruitment of F-actin at cell-cell junctions. We thus propose that Mtss1 promotes Rac1 activation and actin recruitment driving junction maintenance. We suggest that the observed loss of Mtss1 in cancers may compromise junction stability and thus promote EMT and metastasis

Phenotypic and Functional Changes in Blood Monocytes Following Adherence to Endothelium

Blood monocytes are known to express endothelial-like genes during co-culture with endothelium. In this study, the time-dependent change in the phenotype pattern of primary blood monocytes after adhering to endothelium is reported using a novel HLA-A2 mistyped co-culture model.Freshly isolated human PBMCs were co-cultured with human umbilical vein endothelial cells or human coronary arterial endothelial cells of converse human leukocyte antigen A2 (HLA-A2) status. This allows the tracking of the PBMC-derived cells by HLA-A2 expression and assessment of their phenotype pattern over time. PBMCs that adhered to the endothelium at the start of the co-culture were predominantly CD11b+ blood monocytes. After 24 to 72 hours in co-culture, the endothelium-adherent monocytes acquired endothelial-like properties including the expression of endothelial nitric oxide synthase, CD105, CD144 and vascular endothelial growth factor receptor 2. The expression of monocyte/macrophage lineage antigens CD14, CD11b and CD36 were down regulated concomitantly. The adherent monocytes did not express CD115 after 1 day of co-culture. By day 6, the monocyte-derived cells expressed vascular cell adhesion molecule 1 in response to tumour necrosis factor alpha. Up to 10% of the PBMCs adhered to the endothelium. These monocyte-derived cells contributed up to 30% of the co-cultured cell layer and this was dose-dependent on the PBMC seeding density.Human blood monocytes undergo rapid phenotype change to resemble endothelial cells after adhering to endothelium

Angiomotin and angiomotin like proteins, their expression and correlation with angiogenesis and clinical outcome in human breast cancer

BACKGOUND: Angiomotin is a newly discovered molecule that regulates the migration and tubule formation of endothelial cells. It therefore has been implicated in the control of angiogenesis under physiological and pathological conditions. This study examined the expression of angiomotin and its analogues, angiomotin-like 1 (L1) and -like 2 (L2) in breast tumour tissues, and analysed their correlation with angiogenesis and clinical outcomes. METHODS: Human breast tissues (normal n = 32 and tumours n = 120) were used. The levels of expression of angiomotin, L1 and L2 were determined using reverse transcription PCR. Microvessels were stained using antibodies against PECAM, von Willebrand factor (factor 8, or vWF) and VE-cadherin. The transcript levels of angiomotin and its analogues were assessed against the clinical and pathological background, including long term survival (120 months). RESULTS: Breast cancer tissues expressed significantly higher levels of angiomotin transcript, compared with normal mammary tissues (33.1 ± 11 in normal versus 86.5 ± 13.7 in tumour tissues, p = 0.003). Both L1 and L2 were seen at marginally higher levels in tumour than normal tissues but the difference was not statistically significant. Levels of angiomotin were at significantly higher levels in grade 2 and grade 3 tumours compared with grade 1 (p < 0.01 and p = 0.05 respectively). The levels of angiomotin in tumours from patients who had metastatic disease were also significantly higher than those patients who remained disease free (p = 0.03). Multivariate analysis indicated that angiomotin transcript was an independent prognostic factor (p = 0.031). No significant correlations were seen between angiomotin-L1 and L2 with the clinical outcome. Furthermore, high levels of angiomotin transcript were associated with shorter overall survival (p < 0.05). There was a high degree of correlation between levels of vW factor and that of angiomotin (p < 0.05), but not angiomotin-L1 and angiomotin-L2. CONCLUSION: Angiomotin, a putative endothelial motility factor, is highly expressed in human breast tumour tissues and linked to angiogenesis. It links to the aggressive nature of breast tumours and the long term survival of the patients. These data point angiomotin as being a potential therapeutic target

Mesoglycan connects Syndecan-4 and VEGFR2 through Annexin A1 and formyl peptide receptors to promote angiogenesis in vitro.

Mesoglycan is a mixture of glycosaminoglycans (GAG) with fibrinolytic effects and the potential to enhance skin wound repair. Here, we have used endothelial cells isolated from Wild Type (WT) and Syndecan-4 null (Sdc4-/-) C57BL/6 mice to demonstrate that mesoglycan promotes cell motility and in vitro angiogenesis acting on the co-receptor Syndecan-4 (SDC4). This latter is known to participate in the formation and release of extracellular vesicles (EVs). We characterized EVs released by HUVECs and assessed their effect on angiogenesis. Particularly, we focused on Annexin A1 (ANXA1) containing EVs, since they may contribute to tube formation via interactions with Formyl peptide receptors (FPRs). In our model, the bond ANXA1-FPRs stimulates the release of vascular endothelial growth factor (VEGF-A) that interacts with vascular endothelial receptor-2 (VEGFR2) and activates the pathway enhancing cell motility in an autocrine manner, as shown by Wound-Healing/invasion assays, and the induction of Endothelial to Mesenchymal Transition (EndMT). Thus, we have shown for the first time that mesoglycan exerts its pro-angiogenic effects in the healing process triggering the activation of the three interconnected molecular axis: mesoglycan-SDC4, EVs-ANXA1-FPRs and VEGF-A-VEGFR2

Systemic inhibition of tumour angiogenesis by endothelial cell-based gene therapy

Angiogenesis and post-natal vasculogenesis are two processes involved in the formation of new vessels, and both are essential for tumour growth and metastases. We isolated endothelial cells from human blood mononuclear cells by selective culture. These blood outgrowth cells expressed endothelial cell markers and responded correctly to functional assays. To evaluate the potential of blood outgrowth endothelial cells (BOECs) to construct functional vessels in vivo, NOD-SCID mice were implanted with Lewis lung carcinoma cells subcutaneously (s.c.). Blood outgrowth endothelial cells were then injected through the tail vein. Initial distribution of these cells occurred throughout the lung, liver, spleen, and tumour vessels, but they were only found in the spleen, liver, and tumour tissue 48 h after injection. By day 24, they were mainly found in the tumour vasculature. Tumour vessel counts were also increased in mice receiving BOEC injections as compared to saline injections. We engineered BOECs to deliver an angiogenic inhibitor directly to tumour endothelium by transducing them with the gene for human endostatin. These cells maintained an endothelial phenotype and decreased tumour vascularisation and tumour volume in mice. We conclude that BOECs have the potential for tumour-specific delivery of cancer gene therapy

Bone Marrow Derived Mesenchymal Stem Cells Inhibit Inflammation and Preserve Vascular Endothelial Integrity in the Lungs after Hemorrhagic Shock

Hemorrhagic shock (HS) and trauma is currently the leading cause of death in young adults worldwide. Morbidity and mortality after HS and trauma is often the result of multi-organ failure such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), conditions with few therapeutic options. Bone marrow derived mesenchymal stem cells (MSCs) are a multipotent stem cell population that has shown therapeutic promise in numerous pre-clinical and clinical models of disease. In this paper, in vitro studies with pulmonary endothelial cells (PECs) reveal that conditioned media (CM) from MSCs and MSC-PEC co-cultures inhibits PEC permeability by preserving adherens junctions (VE-cadherin and β-catenin). Leukocyte adhesion and adhesion molecule expression (VCAM-1 and ICAM-1) are inhibited in PECs treated with CM from MSC-PEC co-cultures. Further support for the modulatory effects of MSCs on pulmonary endothelial function and inflammation is demonstrated in our in vivo studies on HS in the rat. In a rat “fixed volume” model of mild HS, we show that MSCs administered IV potently inhibit systemic levels of inflammatory cytokines and chemokines in the serum of treated animals. In vivo MSCs also inhibit pulmonary endothelial permeability and lung edema with concurrent preservation of the vascular endothelial barrier proteins: VE-cadherin, Claudin-1, and Occludin-1. Leukocyte infiltrates (CD68 and MPO positive cells) are also decreased in lungs with MSC treatment. Taken together, these data suggest that MSCs, acting directly and through soluble factors, are potent stabilizers of the vascular endothelium and inflammation. These data are the first to demonstrate the therapeutic potential of MSCs in HS and have implications for the potential use of MSCs as a cellular therapy in HS-induced lung injury

Functional characteristics of calcitonin gene-related peptide receptors in human Ewing's sarcoma WE-68 cells

AbstractCalcitonin gene-related peptide (CGRP) receptor activity was studied in WE-68 human Ewing's sarcoma cells. 125I-human CGRP bound in a time-dependent, reversible and saturable manner. Scatchard plots were compatible with the presence of a homogenous population of CGRP receptors with high affinity (Kd = 15 pM, and Bmax = 1.9 fmolmg protein). The potency order of unlabeled peptides, in the presence of radioligand, was: human CGRP-II > human CGRP = chick CGRP > rat CGRP = rat [Tyro]CGRP > human [Tyro] CGRP > > salmon calcitonin (CT) > rat [Tyro]CGRP-(28-37). Each peptide except CT and [Tyio]CGRP-(28-37) stimulated cyclic AMP generation in a concentration-dependent manner, and the relative potencies paralleled their relative ability in inhibiting 125I-human CGRP binding. We conclude that WE-68 Ewing's sarcoma cells express genuine CGRP receptors which upon activation lead to stimulation of cyclic AMP formation.Calcitonin gene-related peptide; Calcitonin; cyclic AMP; (Human; Ewing's sarcoma cell