Transmembrane Protein 184A and Syndecan-4 function in the same pathway to promote angiogenesis

Morpholino (MO) knockdown (KD) of the novel vascular heparin receptor, Transmembrane protein 184A (TMEM184A), increases endothelial cell (EC) proliferation rates, leading to disorganized and stunted vessel outgrowth in the regenerating zebrafish caudal fin. A similar vascular phenotype is observed in the developing zebrafish embryo, where TMEM184A KD reduces the number and length of intersegmental vessels (ISVs), as well as total Vascular Endothelial Cadherin (VEC) protein in ISV ECs. The Heparan Sulfate Proteoglycan Syndecan-4 (Sdcn-4) is required for VEC internalization in angiogenesis and interacts with membrane proteins to promote cellular signaling and molecular processes through interactions with its attached heparan sulfate (HS) chains. Since HS is nearly identical to heparin in structure and both TMEM184A and Sdcn-4 appear to be required for stable angiogenesis, we asked whether TMEM184A and Sdcn-4 interact and function in the same pathway to promote vascular sprouting and regeneration. Using MO knockdown of TMEM184A and Sdcn-4 in the regenerating zebrafish caudal fin, we demonstrate that co-injected subthreshold concentrations of both MOs synergize to produce the same vascular defect. In cells, we find that immunostaining of TMEM184A, Sdcn-4, and VEC show trimeric colocalization at the cell surface, that in vesicle puncta they colocalize with the recycling marker Rab5a, and that TMEM184A precipitates Sdcn-4 in whole cell lysate. In TMEM184A overexpression (OE) backgrounds and in ECs treated with heparin, immunostaining of VEC show increased expression levels in adherens junctions. Collectively, these results suggest that TMEM184A and Sdcn-4 function in the same pathway to promote stable angiogenic repair, that TMEM184A interacts with both Syndecan-4 and VEC in ECs, that they form a complex that is potentially recycled at the membrane surface, and that TMEM184A overexpression or activation stabilizes VEC in adherens junctions. Further study is required to determine whether TMEM184A and Syndecan-4 function together to regulate VEC trafficking and promote angiogenesis or whether TMEM184A expression impacts on VEC membrane stability are distinct from the TMEM184A-Sdcn-4 molecular mechanism that regulates angiogenic repair.