The functional relationship between transglutaminase 2 and transforming growth factor β1 in the regulation of angiogenesis and endothelial-mesenchymal transition

The importance of transglutaminase 2 (TG2) in angiogenesis has been highlighted in recent studies, but other roles of this multi-functional enzyme in endothelial cell (EC) function still remains to be fully elucidated. We previously showed that the extracellular TG2 is involved in maintaining tubule formation in ECs by a mechanism involving matrix-bound vascular endothelial growth factor (VEGF) signalling. Here, by using the ECs and fibroblast co-culture and ECs 3D culture models, we demonstrate a further role for TG2 in both endothelial tubule formation and in tubule loss, which involves its role in the regulation of transforming growth factor β1 (TGFβ1) and Smad signalling. We demonstrate that inhibition of tubule formation by TG2 inhibitors can be restored by add-back of exogenous TGFβ1 at pg/ml levels and show that TG2 -/- mouse ECs are unable to form tubules in 3D culture and display negligible Smad signalling compared to wild-type cells. Loss of tubule formation in the TG2 -/- ECs can be reconstituted by transduction with TG2. We demonstrate that extracellular TG2 also has an important role in TGFβ1-induced transition of ECs into myofibroblast-like cells (endothelial-mesenchymal transition), resulting in loss of EC tubules and tubule formation. Our data also indicate that TG2 may have a role in regulating TGFβ signalling through entrapment of active TGFβ1 into the extracellular matrix. In conclusion, our work demonstrates that TG2 has multi-functional roles in ECs where its ability to fine-tune of TGFβ1 signalling means it can be involved in both endothelial tubule formation and tubule rarefaction

The Lack of ADAM17 Activity during Embryonic Development Causes Hemorrhage and Impairs Vessel Formation

Background: ADAM17/TACE activity is important during embryonic development. We wished to investigate possible roles of this metalloprotease, focusing on vascular development. Methodology/Principal Findings: Mice mutant in the enzymatic activity of ADAM17 were examined at various stages of embryonic development for vascular pattern and integrity using markers for vessel wall cells. We observed hemorrhage and edema starting at embryonic day E14.5 and becoming more severe as development proceeded; prior to embryonic day E14.5, embryos appeared normal. Staining for PECAM-1/CD31 revealed abnormalities in the patterns of branching of the embryonic vasculature at E14.5. Conclusions/Significance: These abnormalities preceded association of pericytes or monocyte/macrophage cells with the affected vessels and, therefore, presumably arise from defects in endothelial function consequent upon failure of ADAM17 to cleave one or more substrates involved in vascular development, such as Notch, Delta, VEGFR2 or JAM-A. Our study demonstrates a role for ADAM17 in modulating embryonic vessel development and function

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background: Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics. Methods and Findings: To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF. Conclusions: These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation

Adhesion Failures Determine the Pattern of Choroidal Neovascularization in the Eye: A Computer Simulation Study

Choroidal neovascularization (CNV) of the macular area of the retina is the major cause of severe vision loss in adults. In CNV, after choriocapillaries initially penetrate Bruch's membrane (BrM), invading vessels may regress or expand (CNV initiation). Next, during Early and Late CNV, the expanding vasculature usually spreads in one of three distinct patterns: in a layer between BrM and the retinal pigment epithelium (sub-RPE or Type 1 CNV), in a layer between the RPE and the photoreceptors (sub-retinal or Type 2 CNV) or in both loci simultaneously (combined pattern or Type 3 CNV). While most studies hypothesize that CNV primarily results from growth-factor effects or holes in BrM, our three-dimensional simulations of multi-cell model of the normal and pathological maculae recapitulate the three growth patterns, under the hypothesis that CNV results from combinations of impairment of: 1) RPE-RPE epithelial junctional adhesion, 2) Adhesion of the RPE basement membrane complex to BrM (RPE-BrM adhesion), and 3) Adhesion of the RPE to the photoreceptor outer segments (RPE-POS adhesion). Our key findings are that when an endothelial tip cell penetrates BrM: 1) RPE with normal epithelial junctions, basal attachment to BrM and apical attachment to POS resists CNV. 2) Small holes in BrM do not, by themselves, initiate CNV. 3) RPE with normal epithelial junctions and normal apical RPE-POS adhesion, but weak adhesion to BrM (e.g. due to lipid accumulation in BrM) results in Early sub-RPE CNV. 4) Normal adhesion of RBaM to BrM, but reduced apical RPE-POS or epithelial RPE-RPE adhesion (e.g. due to inflammation) results in Early sub-retinal CNV. 5) Simultaneous reduction in RPE-RPE epithelial binding and RPE-BrM adhesion results in either sub-RPE or sub-retinal CNV which often progresses to combined pattern CNV. These findings suggest that defects in adhesion dominate CNV initiation and progression