ABSTRACT
BACKGROUND: Thrombospondin type I domain containing 7A (THSD7A) is a novel neural protein that is known to affect endothelial migration and vascular patterning during development. To further understand the role of THSD7A in angiogenesis, we investigated the post-translational modification scheme of THS7DA and to reveal the underlying mechanisms by which this protein regulates blood vessel growth. METHODOLOGY/PRINCIPAL FINDINGS: Full-length THSD7A was overexpressed in human embryonic kidney 293T (HEK293T) cells and was found to be membrane associated and N-glycosylated. The soluble form of THSD7A, which is released into the cultured medium, was harvested for further angiogenic assays. We found that soluble THSD7A promotes human umbilical vein endothelial cell (HUVEC) migration and tube formation. HUVEC sprouts and zebrafish subintestinal vessel (SIV) angiogenic assays further revealed that soluble THSD7A increases the number of branching points of new vessels. Interestingly, we found that soluble THSD7A increased the formation of filopodia in HUVEC. The distribution patterns of vinculin and phosphorylated focal adhesion kinase (FAK) were also affected, which implies a role for THSD7A in focal adhesion assembly. Moreover, soluble THSD7A increased FAK phosphorylation in HUVEC, suggesting that THSD7A is involved in regulating cytoskeleton reorganization. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that THSD7A is a membrane-associated N-glycoprotein with a soluble form. Soluble THSD7A promotes endothelial cell migration during angiogenesis via a FAK-dependent mechanism and thus may be a novel neuroangiogenic factor.
Subject(s)
Cell Movement , Glycoproteins/metabolism , Human Umbilical Vein Endothelial Cells/cytology , Neovascularization, Physiologic , Thrombospondins/metabolism , Animals , Antibodies, Blocking/pharmacology , Blood Vessels/drug effects , Blood Vessels/growth & development , Blood Vessels/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Movement/drug effects , Embryo, Nonmammalian/blood supply , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Extracellular Space/drug effects , Extracellular Space/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Focal Adhesions/drug effects , Focal Adhesions/metabolism , Glycoproteins/chemistry , Glycosylation/drug effects , HEK293 Cells , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/enzymology , Humans , Intestines/blood supply , Intestines/drug effects , Models, Biological , Neovascularization, Physiologic/drug effects , Protein Binding/drug effects , Protein Structure, Tertiary , Pseudopodia/drug effects , Pseudopodia/metabolism , Solubility/drug effects , Thrombospondins/chemistry , Zebrafish/embryology , Zebrafish/metabolismABSTRACT
Angiogenesis is a highly organized process under the control of guidance cues that direct endothelial cell (EC) migration. Recently, many molecules that were initially described as regulators of neural guidance were subsequently shown to also direct EC migration. Here, we report a novel protein, thrombospondin type I domain containing 7A (Thsd7a), that is a neural molecule required for directed EC migration during embryonic angiogenesis in zebrafish. Thsd7a is a vertebrate conserved protein. Zebrafish thsd7a transcript was detected along the ventral edge of the neural tube in the developing zebrafish embryos, correlating with the growth path of angiogenic intersegmental vessels (ISVs). Morpholino-knockdown of Thsd7a caused a lateral deviation of angiogenic ECs below the thsd7a-expressing sites, resulting in aberrant ISV patterning. Collectively, our study shows that zebrafish Thsd7a is a neural protein required for ISV angiogenesis, and suggests an important role of Thsd7a in the neurovascular interaction during zebrafish development.
Subject(s)
Blood Vessels/embryology , Body Patterning/genetics , Neovascularization, Physiologic/genetics , Thrombospondins/physiology , Zebrafish Proteins/physiology , Zebrafish/embryology , Amino Acid Sequence , Animals , Animals, Genetically Modified , Blood Vessels/metabolism , Central Nervous System/embryology , Central Nervous System/metabolism , Embryo, Nonmammalian , Molecular Sequence Data , Neovascularization, Physiologic/physiology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/physiology , Phylogeny , Sequence Homology, Amino Acid , Thrombospondins/genetics , Thrombospondins/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
In this work, the zebrafish model organism was developed to obtain a minivertebrate host system for a Candida albicans infection study. We demonstrated that C. albicans can colonize and invade zebrafish at multiple anatomical sites and kill the fish in a dose-dependent manner. Inside zebrafish, we monitored the progression of the C. albicans yeast-to-hypha transition by tracking morphogenesis, and we monitored the corresponding gene expression of the pathogen and the early host immune response. We performed a zebrafish survival assay with different C. albicans strains (SC5314, ATCC 10231, an hgc1 mutant, and a cph1/efg1 double mutant) to determine each strain's virulence, and the results were similar to findings reported in previous mouse model studies. Finally, using zebrafish embryos, we monitored C. albicans infection and visualized the interaction between pathogen and host myelomonocytic cells in vivo. Taken together, the results of this work demonstrate that zebrafish can be a useful host model to study C. albicans pathogenesis, and they highlight the advantages of using the zebrafish model in future invasive fungal research.
