Thromboxane Mobilizes Insect Blood Cells to Infection Foci

Innate immune responses are effective for insect survival to defend against entomopathogens including a fungal pathogen, Metarhizium rileyi, that infects a lepidopteran Spodoptera exigua. In particular, the fungal virulence was attenuated by cellular immune responses, in which the conidia were phagocytosed by hemocytes (insect blood cells) and hyphal growth was inhibited by hemocyte encapsulation. However, the chemokine signal to drive hemocytes to the infection foci was little understood. The hemocyte behaviors appeared to be guided by a Ca2+ signal stimulating cell aggregation to the infection foci. The induction of the Ca2+ signal was significantly inhibited by the cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A2 or B2 (TXA2 or TXB2) among COX products was the most effective to recover the Ca2+ signal and hemocyte aggregation. TXB2 alone induced a microaggregation behavior of hemocytes under in vitro conditions. Indeed, TXB2 titer was significantly increased in the plasma of the infected larvae. The elevated TXB2 level was further supported by the induction of phospholipase A2 (PLA2) activity in the hemocytes and subsequent up-regulation of COX-like peroxinectins (SePOX-F and SePOX-H) in response to the fungal infection. Finally, the expression of a thromboxane synthase (Se-TXAS) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited the Ca2+ signal and hemocyte aggregation around fungal hyphae, which were rescued by the addition of TXB2. Without any ortholog to mammalian thromboxane receptors, a prostaglandin receptor was essential to mediate TXB2 signal to elevate the Ca2+ signal and mediate hemocyte aggregation behavior. Specific inhibitor assays suggest that the downstream signal after binding TXB2 to the receptor follows the Ca2+-induced Ca2+ release pathway from the endoplasmic reticulum of the hemocytes. These results suggest that hemocyte aggregation induced by the fungal infection is triggered by TXB2via a Ca2+ signal through a PG receptor.

Screening of Synthetic Heterocyclic Compounds as Antiplatelet Drugs

Background: Antiplatelet drugs represent the keystone in the treatment and prevention of diseases of ischemic origin, including coronary artery disease. The current palette of drugs represents efficient modalities in most cases, but their effect can be limited in certain situations or associated with specific side effects. In this study, representatives of compounds selected from series having scaffolds with known or potential antiplatelet activity were tested. These compounds were previously synthetized by us, but their biological effects have not yet been reported. Objective: The aim of this study was to examine the antiplatelet and anticoagulation properties of selected compounds and determine their mechanism of action. Methods: Antiplatelet activity of compounds and their mechanisms of action were evaluated using human blood by impedance aggregometry and various aggregation inducers and inhibitors and compared to appropriate standards. Cytotoxicity was tested using breast adenocarcinoma cell cultures and potential anticoagulation activity was also determined. Results: In total, four of 34 compounds tested were equally or more active than the standard antiplatelet drug acetylsalicylic acid (ASA). In contrast to ASA, all 4 active compounds decreased platelet aggregation triggered not only by collagen, but also partly by ADP. The major mechanism of action is based on antagonism at thromboxane receptors. In higher concentrations, inhibition of thromboxane synthase was also noted. In contrast to ASA, the tested compounds did not block cyclooxygenase-1. Conclusion: The most active compound, 2-amino-4-(1H-indol-3-yl)-6-nitro-4H-chromene-3-carbonitrile (2-N), which is 4-5x times more potent than ASA, is a promising compound for the development of novel antiplatelet drugs.

Abstract 133: Mechanistic Insights Into Bradykinin and Thromboxane Receptors Heterodimerization in Vascular Smooth Muscle Cells

The development of an atherosclerotic lesion in an injured vasculature is highly dependent on the proliferative state of vascular smooth muscle cells (VSMC). Bradykinin (BK) and Thromboxane are two G-protein coupled receptor ligands, whose individual binding to their respective receptors, B 2 R and TP, promotes ERK1/2-mediated VSMC proliferation. However, it is not yet known whether receptor-receptor interactions between B 2 R and TP could contribute to their co-regulation. Thus, our work addresses the hypothesis that, in VSMC, B 2 R and TP form functional hetero-complexes, of distinct trafficking and signaling properties. B 2 R-TP signaling crosstalk was first analyzed in rat VSMC by Western Blot analysis and subsequent fold/basal quantification of ERK1/2 phosphorylation. B 2 R-TP cooperation was evident through the synergistic ERK1/2 phosphorylation, in VSMC co-stimulated with optimized combinations of BK and the TP agonist, IBOP (21.05 ± 4.93 fold of basal, n=3; p < 0.001). Interestingly, however, pretreatment with the TP antagonist, SQ29548, totally abolished (BK+IBOP)-induced ERK1/2 (n=3; p < 0.001). Furthermore, knowing that B 2 R, unlike the human TPα isoform, undergoes agonist-induced sequestration and β-arrestin2 recruitment, we conducted immunofluorescence analysis on HEK293T cells overexpressing human B 2 R and human TPα (B 2 R-TPα-HEKs). While stimulation with IBOP failed to mobilize cell membranous B 2 R or TPα, substantial co-internalization of TPα and B 2 R was seen, subsequent to stimulation of B 2 R-TPα-HEKs with BK. Likewise, overexpressing β-arrestin2 in B 2 R-TPα-HEKs resulted in co-localization of B 2 R, TPα, and β-arrestin2 within internalized puncta, only after BK stimulation. Pretreatment with SQ29548 inhibited BK-induced co-internalization and β-arrestin2 recruitment. However, SQ29548 could not reverse BK-induced B 2 R sequestration in HEK293T cells overexpressing B 2 R alone, thus excluding the possibility of direct SQ29548 - B 2 R binding. Finally, results of our ongoing work are the first to show nuclear localization of B 2 R and TP within human and rat VSMC. Thus, our findings favor the likelihood of functional B 2 R-TP heterodimerization in VSMC, which could serve as a novel target for interventional strategies.