ABSTRACT
BACKGROUND: Endothelial cells provide a barrier between blood and tissues, which is regulated to allow molecules and cells in out of tissues. Patients with cerebral cavernous malformations (CCM) have dilated leaky blood vessels, especially in the central nervous system. A subset of these patients has loss-of-function mutations in CCM3. CCM3 is part of the STRIPAK protein complex that includes the little-characterized proteins FAM40A and FAM40B. RESULTS: We show here that FAM40A and FAM40B can interact with CCM3. Knockdown of CCM3, FAM40A or FAM40B in endothelial cells by RNAi causes an increase in stress fibers and a reduction in loop formation in an in vitro angiogenesis assay, which can be reverted by inhibiting the Rho-regulated ROCK kinases. FAM40B depletion also increases endothelial permeability. CONCLUSIONS: These results demonstrate the importance of the FAM40 proteins for endothelial cell physiology, and suggest that they act as part of the CCM3-containing STRIPAK complex.
Subject(s)
Carrier Proteins/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , rho-Associated Kinases/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , COS Cells , Cell Membrane Permeability , Chlorocebus aethiops , Cytoskeletal Proteins , Humans , Membrane Proteins/metabolism , Myosin Light Chains/metabolism , Neovascularization, Physiologic , Phosphate-Binding Proteins , Phosphorylation , Proto-Oncogene Proteins/metabolism , Signal Transduction , Stress Fibers/metabolism , rho GTP-Binding Proteins/metabolismABSTRACT
A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15 was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting.
Subject(s)
ADAM Proteins/metabolism , Amyloid Precursor Protein Secretases/metabolism , Blood Platelets/metabolism , Cell Membrane/metabolism , Endothelium, Vascular/metabolism , Membrane Proteins/metabolism , Tetraspanins/metabolism , ADAM Proteins/chemistry , ADAM Proteins/genetics , ADAM10 Protein , Amyloid Precursor Protein Secretases/chemistry , Amyloid Precursor Protein Secretases/genetics , Animals , Blood Platelets/cytology , Cell Line , Cell Membrane/enzymology , Cells, Cultured , Endothelium, Vascular/cytology , Enzyme Activation , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Protein Transport , Proteolysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Surface Properties , Tetraspanin 29/chemistry , Tetraspanin 29/genetics , Tetraspanin 29/metabolism , Tetraspanins/chemistry , Tetraspanins/geneticsABSTRACT
Cell therapies to treat critical limb ischaemia have demonstrated only modest results in clinical trials, and this has been partly attributed to poor cell retention following their delivery directly into the ischaemic limb. The aim of this study was to determine whether alginate encapsulation of therapeutic pro-angio/arteriogenic macrophages enhances their retention and ultimately improves limb perfusion. A reproducible GMP-compliant method for generating 300 µm alginate capsules was developed to encapsulate pro-angio/arteriogenic macrophages. Longitudinal analysis revealed no detrimental effect of encapsulation on cell number or viability in vitro, and macrophages retained their pro-angio/arteriogenic phenotype. Intramuscular delivery of encapsulated macrophages into the murine ischaemic hindlimb demonstrated increased cell retention compared with injection of naked cells (P = 0.0001), and that this was associated both enhanced angiogenesis (P = 0.02) and arteriogenesis (P = 0.03), and an overall improvement in limb perfusion (P = 0.0001). Alginate encapsulation of pro-angio/arteriogenic macrophages enhances cell retention and subsequent limb reperfusion in vivo. Encapsulation may therefore represent a means of improving the efficacy of cell-based therapies currently under investigation for the treatment of limb ischaemia.