RhoJ/TCL Regulates Endothelial Motility and Tube Formation and Modulates Actomyosin Contractility and Focal Adhesion Numbers

Objective—RhoJ/TCL was identified by our group as an endothelial-expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation. Methods and Results—Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence. The proangiogenic factor vascular endothelial growth factor activated RhoJ in endothelial cells. Using either small interfering (si)RNA-mediated knockdown of RhoJ expression or overexpression of constitutively active RhoJ (daRhoJ), RhoJ was found to positively regulate endothelial motility and tubule formation. Downregulating RhoJ expression increased focal adhesions and stress fibers in migrating cells, whereas daRhoJ overexpression resulted in the converse. RhoJ downregulation resulted in increased contraction of a collagen gel and increased phospho–myosin light chain, indicative of increased actomyosin contractility. Pharmacological inhibition of Rho-kinase (which phosphorylates myosin light chain) or nonmuscle myosin II reversed the defective tube formation and migration of RhoJ knockdown cells. Conclusion—RhoJ is endothelial-expressed in vivo, activated by vascular endothelial growth factor, localizes to focal adhesions, regulates endothelial cell migration and tube formation, and modulates actomyosin contractility and focal adhesion numbers

Brain endothelial cells promote breast cancer cell extravasation to the brain via EGFR-DOCK4-RAC1 signalling

Peer reviewe

Chemically-induced Neurite-like Outgrowth Reveals Multicellular Network Function in Patient-derived Glioblastoma Cells

Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of Glioblastoma Multiforme (GBM), the most aggressive type of brain cancer in adults. We show that small molecule inhibition of RHO-associated serine/threonine kinase (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed de novo -induced cellular network (iNet) ‘webs’, which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range calcium signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs ex vivo , and may thereby accelerate future studies into the biology of GBM cellular networks

Scleroderma fibroblasts suppress angiogenesis via TGF-β/caveolin-1 dependent secretion of pigment epithelium-derived factor

Objectives: Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. Methods: PEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast–endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis. Results: PEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. Conclusions: Our data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc

Uses of the in vitro endothelial-fibroblast organotypic co-culture assay in angiogenesis research

Angiogenesis is a complex process that involves multiple cellular events. In addition to receiving inputs from a range of stimulatory and inhibitory factors, endothelial cells undergoing angiogenesis make multiple interactions with the extracellular matrix and with other cell types in the stroma. Recreating angiogenesis in vitro is probably an impossible goal; however, a number of assays have been developed that recapitulate many of the key events of the process. These assays are indispensible tools for investigating the signalling pathways that control the formation of new blood vessels. In the present paper, we review the organotypic co-culture assay of angiogenesis – until recently, a comparatively underemployed assay, but one with a number of powerful advantages for angiogenesis research. We give a set of optimized protocols for its use, including protocols for siRNA (small interfering RNA)-based screens, and we discuss appropriate methods for obtaining quantitative data from the assay.</jats:p

VE-Cadherin-Mediated Cell-Cell Interaction Suppresses Sprouting via Signaling to MLC2 Phosphorylation

SummaryDuring new blood vessel formation, the cessation of angiogenic sprouting is necessary for the generation of functional vasculature. How sprouting is halted is not known, but it is contemporaneous with the development of stable intercellular junctions [1]. We report that VE-cadherin, which is responsible for endothelial adherens junction organization [2, 3], plays a crucial role in the cessation of sprouting. Abrogating VE-cadherin function in an organotypic angiogenesis assay and in zebrafish embryos stimulates sprouting. We show that VE-cadherin signals to Rho-kinase-dependent myosin light-chain 2 phosphorylation, leading to actomyosin contractility [4], which regulates the distribution of VE-cadherin at cell-cell junctions. VE-cadherin antagonizes VEGFR2 signaling, and consequently, inhibition of VE-cadherin, Rho-kinase, or actomyosin contractility leads to VEGF-driven, Rac1-dependent sprouting. These findings suggest a novel mechanism by which cell-cell adhesion suppresses Rac1-dependent migration and sprouting by increasing actomyosin contractility at cell junctions

Targeting Tumour Vasculature using Integrin αvβ3 -Observation of Liposome Accumulation in Microfluidic Vasculature Networks

This paper reports the use of perfusable microfluidic vasculature networks to investigate the use αvβ3 integrin-targeted liposomes as tumour vasculature targeting agents. Endothelial cells co-cultured with fibroblasts were induced to self-assemble into vasculature networks using pro-angiogenic growth factors. Tumour vasculature was mimicked by growing networks in tumour-cell conditioned media (TCM), taken from HCT116 cell cultures. Assessment of αvβ3 expression using immunostaining and flow cytometry observed the upregulation of αvβ3 expression on endothelial cells grown in TCM. Results observed increased liposome accumulation in tumour conditioned networks, suggesting αvβ3 may be a promising target for anti-cancer therapeutics