Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparininduced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNF␣-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNF␣-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.For almost 100 years heparin has been used as an anticoagulant. Specific heparin interactions with proteins important in the anti-clotting system are now well understood. Many heparin binding proteins, including quite a few involved in modulating vascular function, inflammation, and angiogenesis, have been identified (reviewed in Ref. 1). The large number of reports indicating evidence of decreased endogenous heparin and heparan sulfates (HS) 3 in atherosclerosis (in model animals and human disease) led to a proposal that decreases in endogenous heparins might be important in the development of atherosclerosis (2). More recent evidence in support of that hypothesis includes increased heparanase expression in atherosclerosis (reviewed in Ref.3) and increased levels of glycocalyx heparan sulfate in regions of the vasculature where laminar flow decreases the likelihood of atherosclerosis development (4).In addition to heparin, heparin binding proteins typically also bind HS chains on HS proteoglycans (HSPGs). Although the carbohydrate backbones of heparin and HS are identical, modifications and sulfation patterns vary. HS chains have fewer sulfate residues per disaccharide and a lower overall charge, but their widespread expression suggests that HS may provide many in vivo functions identified originally as heparin functions (reviewed in Ref. 5). Heparin binding to growth factors modulates their activity and appears to protect them from degradation...

It has become increasingly clear that stress-activated protein kinases have cytoplasmic substrates in addition to well-established transcription factor substrates in cell nuclei. The present study documented specific cytoplasmic locations of these enzymes in proliferating vascular cells. Immunofluorescent staining for active c-jun NH2-terminal kinase (JNK), the precipitation of JNK with microfilaments, and the loss of fiber-associated active JNK after cytochalasin treatment, but not nocodazole treatment, together indicate that active JNK is associated with stress fibers. The lack of complete scaffold colocalization and the total lack of immediate upsteam kinase colocalization along with the inability of JNK inhibitors to alter JNK-microfilament associations suggest that the microfilament association is not simply involved in enzyme activation. In addition, active p38 was found along with vinculin in focal adhesions. Although the p38 in focal adhesions could also be disrupted by cytochalasin treatment, it remained stable after nocodazole treatment. These results support the hypothesis that vascular cell stress kinase enzymes are important for signal transduction in the cytoplasm. The localization of active stress-activated protein kinases to specific cytoskeletal structures in proliferating cells suggests that subsets of these enzymes are involved in signal transduction to and/or from the cytoskeleton under conditions that include vascular cell proliferation.