Reprogramming Megakaryocytes for Controlled Release of Platelet-like Particles Carrying a Single-Chain Thromboxane A2 Receptor-G-Protein Complex with Therapeutic Potential.

In this study, we reported that novel single-chain fusion proteins linking thromboxane A2 (TXA2) receptor (TP) to a selected G-protein α-subunit q (SC-TP-Gαq) or to α-subunit s (SC-TP-Gαs) could be stably expressed in megakaryocytes (MKs). We tested the MK-released platelet-linked particles (PLPs) to be used as a vehicle to deliver the overexpressed SC-TP-Gαq or the SC-TP-Gαs to regulate human platelet function. To understand how the single-chain TP-Gα fusion proteins could regulate opposite platelet activities by an identical ligand TXA2, we tested their dual functions-binding to ligands and directly linking to different signaling pathways within a single polypeptide chain-using a 3D structural model. The immature MKs were cultured and transfected with cDNAs constructed from structural models of the individual SC-TP-Gαq and SC-TP-Gαs, respectively. After transient expression was identified, the immature MKs stably expressing SC-TP-Gαq or SC-TP-Gαs (stable cell lines) were selected. The stable cell lines were induced into mature MKs which released PLPs. Western blot analysis confirmed that the released PLPs were carrying the recombinant SC-TP-Gαq or SC-TP-Gαs. Flow cytometry analysis showed that the PLPs carrying SC-TP-Gαq were able to perform the activity by promoting platelet aggregation. In contrast, PLPs carrying SC-TP-Gαs reversed Gq to Gs signaling to inhibit platelet aggregation. This is the first time demonstrating that SC-TP-Gαq and SC-TP-Gαs were successfully overexpressed in MK cells and released as PLPs with proper folding and programmed biological activities. This bio-engineering led to the formation of two sets of biologically active PLP forms mediating calcium and cAMP signaling, respectively. As a result, these PLPs are able to bind to identical endogenous TXA2 with opposite activities, inhibiting and promoting platelet aggregation as reprogrammed for therapeutic process. Results also demonstrated that the nucleus-free PLPs could be used to deliver recombinant membrane-bound GPCRs to regulate cellular activity in general.

Latest advancements in the study of the relationship between NSAIDs and three prostaglandin E2 synthases.

Tweetable abstract This work describes novel evidence of the relationship between NSAIDs and three prostaglandin E2 synthases.

Design, synthesis and characterization of lead compounds as anti-inflammatory drugs targeting mPGES-1 via enzymelink screening.

Aim: The objective of this study was to synthesize and validate a set of compounds that selectively inhibit mPGES-1, with the potential to be developed into a novel anti-inflammatory drug. Methods: The synthesized compounds were characterized using 1H NMR spectroscopy and LC-MS to confirm their structure. Cellular and enzymatic assays were used to demonstrate their inhibitory activity on prostaglandin E2 production. Results: Docking studies revealed that compounds containing fluoro-, chloro- and methyl- groups displayed strong inhibitory activity against prostaglandin E2. The inhibitory activity of synthesized trimethyl and trifluoro was further validated using enzymatic and cell migration assays. Conclusion: The findings demonstrated that the synthesized compounds possess significant potential as a new generation of nonsteroidal anti-inflammatory drugs that selectively target mPGES-1 with fewer side effects.

Engineering 'Enzymelink' for screening lead compounds to inhibit mPGES-1 while maintaining prostacyclin synthase activity.

Aim: This study investigated our Enzymelinks, COX-2-10aa-mPGES-1 and COX-2-10aa-PGIS, as cellular cross-screening targets for quick identification of lead compounds to inhibit inflammatory PGE2 biosynthesis while maintaining prostacyclin synthesis. Methods: We integrated virtual and wet cross-screening using Enzymelinks to rapidly identify lead compounds from a large compound library. Results: From 380,000 compounds virtually cross-screened with the Enzymelinks, 1576 compounds were identified and used for wet cross-screening using HEK293 cells that overexpressed individual Enzymelinks as targets. The top 15 lead compounds that inhibited mPGES-1 activity were identified. The top compound that specifically inhibited inflammatory PGE2 biosynthesis alone without affecting COX-2 coupled to PGI2 synthase (PGIS) for PGI2 biosynthesis was obtained. Conclusion: Enzymelink technology could advance cyclooxygenase pathway-targeted drug discovery to a significant degree.

Suppression of microRNA-16 protects against acute myocardial infarction by reversing beta2-adrenergic receptor down-regulation in rats.

microRNA-16 (miR-16) has been shown to be up-regulated in ischemic heart. Beta2-adrenoreceptor (ß2-AR) exerts cardioprotective property in ischemic injury. This study aims to determine the effect of miR-16 in cardiac injury in rats and the possible involvement of ß2-AR in this process. Acute myocardial infarction (AMI) model in rats was induced by ligation of left coronary artery. Neonatal rat ventricular cells (NRVCs) were cultured in vitro tests. The cardiomyocyte model of oxidative injury was mimicked by hydrogen peroxide. The expression of miR-16 was obviously up-regulated and ß2-AR was remarkably down-regulated in both AMI rats and NRVCs under oxidative stress. miR-16 over-expression in NRVCs reduced cell viability and increased apoptosis. Conversely, inhibition of endogenous miR-16 with its specific inhibitor reversed these changes. Over-expression of miR-16 using an miR-16 lentivirus in AMI rats markedly increased cardiac infarct area, lactate dehydrogenase and creatine kinase activity, and exacerbated cardiac dysfunction. Lentivirus-mediated knockdown of miR-16 alleviated acute cardiac injury. Moreover, miR-16 over-expression significantly suppressed ß2-AR protein expression in both cultured NRVCs and AMI rats, while inhibition of miR-16 displayed opposite effect on ß2-AR protein expression. Luciferase assay confirmed that miR-16 could directly target the 3'untranslated region of ß2-AR mRNA. miR-16 is detrimental to the infarct heart and suppression of miR-16 protects rat hearts from ischemic injury via up-regulating of ß2-AR by binding to the 3'untranslated region of ß2-AR gene. This study indicates that targeting miR-16/ß2-AR axis may be a promising strategy for ischemic heart disease.

Let-7a Is an Antihypertrophic Regulator in the Heart via Targeting Calmodulin.

Background: MicroRNAs (miRNAs) have been emerged as important regulator in a multiple of cardiovascular disease, including arrhythmia, cardiac hypertrophy and fibrosis, and myocardial infarction. The aim of this study was to investigate whether miRNA let-7a has antihypertrophic effects in angiotensin II (AngII)-induced cardiac hypertrophy. Methods: Neonatal rat ventricular myocytes (NRVMs) were exposed to AngII for 36 h as a cellular model of hypertrophy; subcutaneous injection of AngII for 2 weeks was used to establish a mouse model of cardiac hypertrophy in vivo study. Cell surface area (CSA) was measured by immunofluorescence cytochemistry; expression of hypertrophy-related genes ANP, BNP, ß-MHC was detected by Real-time PCR; luciferase activity assay was performed to confirm the miRNA's binding site in the calmodulin (CaM) gene; CaM protein was detected by Western blot; the hypertrophy parameters were measured by echocardiographic assessment. Results: The expression of let-7a was decreased in AngII-induced cardiac hypertrophy in vitro and in vivo. Overexpression of let-7a attenuated AngII-induced increase of cell surface area and repressed the increased mRNA levels of ANP, BNP and ß-MHC. Dual-luciferase reporter assay showed that let-7a could bind to the 3'UTR of CaM 1 gene. Let-7a downregulated the expression of CaM protein. In vivo, let-7a produced inhibitory effects on cardiac hypertrophy, including the downregulation of cross-sectional area of cardiomyocytes in mouse heart, the reduction of IVSD and LVPWD, the suppression of hypertrophy marker genes ANP, BNP, ß-MHC mRNA level, and the downregulation of CaM protein level. Conclusions: let-7a possesses a prominent anti-hypertrophic property by targeting CaM genes. The findings provide new insight into molecular mechanism of cardiac hypertrophy.

Cholesterol Retards Senescence in Bone Marrow Mesenchymal Stem Cells by Modulating Autophagy and ROS/p53/p21Cip1/Waf1 Pathway.

In the present study, we demonstrated that bone marrow mesenchymal stem cells (BMSCs) of the 3rd passage displayed the senescence-associated phenotypes characterized with increased activity of SA-ß-gal, altered autophagy, and increased G1 cell cycle arrest, ROS production, and expression of p53 and p21Cip1/Waf1 compared with BMSCs of the 1st passage. Cholesterol (CH) reduced the number of SA-ß-gal positive cells in a dose-dependent manner in aging BMSCs induced by H2O2 and the 3rd passage BMSCs. Moreover, CH inhibited the production of ROS and expression of p53 and p21Cip1/Waf1 in both cellular senescence models and decreased the percentage of BMSCs in G1 cell cycle in the 3rd passage BMSCs. CH prevented the increase in SA-ß-gal positive cells induced by RITA (reactivation of p53 and induction of tumor cell apoptosis, a p53 activator) or 3-MA (3-methyladenine, an autophagy inhibitor). Our results indicate that CH not only is a structural component of cell membrane but also functionally contributes to regulating cellular senescence by modulating cell cycle, autophagy, and the ROS/p53/p21Cip1/Waf1 signaling pathway.

Expression signature of lncRNAs and their potential roles in cardiac fibrosis of post-infarct mice.

The present study aimed to investigate whether long non-coding RNAs (lncRNAs) are involved in cardiac fibrogenesis induced by myocardial infarction (MI). The differentially expressed lncRNAs and mRNAs in peri-infarct region of mice 4 weeks after MI were selected for bioinformatic analysis including gene ontology (GO) enrichment, pathway and network analysis. Left ventricular tissue levels of lncRNAs and mRNAs were compared between MI and sham control mice, using a false discovery rate (FDR) of <5%. Out of 55000 lncRNAs detected, 263 were significantly up-regulated and 282 down-regulated. Out of 23000 mRNAs detected, 142 were significantly up-regulated and 67 down-regulated. Among the differentially expressed lncRNAs, 53 were up-regulated by ≥2.0-fold change and 37 down-regulated by ≤0.5-fold change. Nine up-regulated and five down-regulated lncRNAs were randomly selected for quantitative real-time PCR (qRT-PCR) verification. GO and pathway analyses revealed 173 correlated lncRNA-mRNA pairs for 57 differentially expressed lncRNAs and 20 differentially expressed genes which are related to the development of cardiac fibrosis. We identified TGF-ß3 as the top-ranked gene, a critical component of the transforming growth factor-ß (TGF-ß) and mitogen activated protein kinase (MAPK) signalling pathways in cardiac fibrosis. NONMMUT022554 was identified as the top-ranked lncRNA, positively correlated with six up-regulated genes, which are involved in the extracellular matrix (ECM)-receptor interactions and the phosphoinositid-3 kinase/protein kinase B (PI3K-Akt) signalling pathway. Our study has identified the expression signature of lncRNAs in cardiac fibrosis induced by MI and unravelled the possible involvement of the deregulated lncRNAs in cardiac fibrosis and the associated pathological processes.