Heparin's Anti‐Inflammatory Effects in Vascular Endothelial Cells are Mediated Through Transmembrane Receptor 184A

Abstract

The glycosaminoglycan heparin has been used as an anti‐coagulant in the treatment of cardiovascular diseases for decades. The mechanism of heparin binding to enzyme inhibitor antithrombin III in the coagulation cascade has been well‐documented. Heparin has also been shown to illicit anti‐proliferative and anti‐inflammatory responses in vascular cells, though mechanism(s) remain unclear. Recently we have used antibodies that mimic heparin effects in vascular smooth muscle cells to identify transmembrane protein 184A (TMEM184A) as a putative heparin receptor. The objective of this study was to investigate TMEM184A expression and function in vascular endothelial cells. We observed TMEM184A expression in cultured endothelial cells and in the zebrafish caudal fin vasculature through immunofluorescence using commercial antibodies against a region near the N‐terminus and a region of 20 amino acids of an internal region. To determine whether heparin effects on inflammatory responses were mediated through TMEM184A, rat aortic endothelial cells (RAOECs) were electroporated with control or TMEM184A shRNA. Control and knockdown RAOECs were treated with 200 μg/ml heparin for 20 min, followed by 25 ng/mL TNFα. Quantitative immunofluorescence of activated JNK or p38 showed that heparin treatment prior to TNFα significantly decreased JNK and p38 activation in control shRNA cells compared to cells treated with TNFα alone. However, in TMEM184A knockdown cells, heparin pre‐treatment did not decrease JNK or p38 activation. Heparin induction of DUSP1 was found to be critical for these effects. In control ECs, heparin pre‐treatment decreased TNFα‐induced stress fiber formation. To investigate whether TMEM184A was also necessary for these effects, stress fibers were quantified through immunofluorescence. In TMEM184A knockdown RAOECs, heparin pre‐treatment did not decrease TNFα‐induced actin stress fiber formation. In conclusion, our results show that TMEM184A is expressed specifically in the vasculature in vitro and in vivo. We provide functional evidence that anti‐inflammatory effects of heparin are dependent at least in part on TMEM184A. Further investigation of heparin's anti‐inflammatory effects through TMEM184A in the vasculature is crucial for developing and improving therapeutic strategies in vascular disease.Support or Funding InformationThis work was supported by NIH grant HL54269 to LLK