RESUMO
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.
Assuntos
Movimento Celular , Glicoproteínas/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Neovascularização Fisiológica , Trombospondinas/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Glicoproteínas/química , Glicosilação/efeitos dos fármacos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Intestinos/irrigação sanguínea , Intestinos/efeitos dos fármacos , Modelos Biológicos , Neovascularização Fisiológica/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Solubilidade/efeitos dos fármacos , Trombospondinas/química , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Vasos Sanguíneos/embriologia , Padronização Corporal/genética , Neovascularização Fisiológica/genética , Trombospondinas/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Vasos Sanguíneos/metabolismo , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Embrião não Mamífero , Dados de Sequência Molecular , Neovascularização Fisiológica/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Filogenia , Homologia de Sequência de Aminoácidos , Trombospondinas/genética , Trombospondinas/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.