Establishing novel prostacyclin-synthesizing cells with therapeutic potential against heart diseases.

BACKGROUND: For decades, there have been many ongoing attempts to use prostaglandin I(2) (PGI(2)) to treat heart diseases, such as pulmonary arterial hypertension. However, the short half life of PGI(2) has limited the therapeutic impact potential. METHODS: Here, we have engineered a novel adipose tissue-derived cell that constantly produces PGI(2,) through transfecting of an engineered cDNA of a hybrid enzyme (human COX-1-10-aa-PGIS) which has superior triple catalytic functions in directly converting arachidonic acid into PGI(2). RESULTS: The gene-transfected cells were further converted into a stable cell line, in which cells constantly express the hybrid enzyme and are capable of producing large-amounts of PGI(2). In a comparison between un-transfected- and gene-transfected cells, it was determined that the majority of the endogenous AA metabolism shifted from that of unwanted PGE(2) (in un-transfected cells) to that of the preferred PGI(2) (in gene-transfected cells) with a PGI(2)/PGE(2) ratio change from 0.03 to 25. The PGI(2)-producing cell line not only exhibited an approximate 50-fold increase in PGI(2) biosynthesis, but also demonstrated superior anti-platelet aggregation in vitro, and increased reperfusion in the mouse ischemic hindlimb model in vivo. CONCLUSIONS: The cells, which have an ability to increase the biosynthesis of the vascular protector, PGI(2), while reducing that of the vascular inflammatory mediator, PGE(2), provide a dual effect on vascular protection, which is not available through any existing drug treatments. Thus, the current finding has potential to be an experimental intervention for PGI(2)-deficient heart diseases, such as pulmonary arterial hypertension.

Inducible COX-2 dominates over COX-1 in prostacyclin biosynthesis: mechanisms of COX-2 inhibitor risk to heart disease.

AIM: Our aim is to understand the molecular mechanisms of the selective nonsteroidal anti-inflammatory drugs (NSAID), cyclooxygenase-2 (COX-2) inhibitors', higher "priority" to reduce synthesis of the vascular protector, prostacyclin (PGI2), compared to that of nonselective NSAIDs. MAIN METHODS: COX-1 or COX-2 was co-expressed with PGI2 synthase (PGIS) in COS-7 cells. The Km and initial velocity (½t Vmax) of the coupling reaction between COX-1 and COX-2 to PGIS were established. The experiment was further confirmed by a kinetics study using hybrid enzymes linking COX-1 or COX-2 to PGIS. Finally, COX-1 or COX-2 and PGIS were respectively fused to red (RFP) and cyanic (CFP) fluorescence proteins, and co-expressed in cells. The distances between COXs and PGIS were compared by FRET. KEY FINDINGS: The Km for converting arachidonic acid (AA) to PGI2 by COX-2 coupled to PGIS is ~2.0µM; however, it was 3-fold more (~6.0µM) for COX-1 coupled to PGIS. The Km and ½t Vmax for COX-2 linked to PGIS were ~2.0µM and 20s, respectively, which were 2-5 folds faster than that of COX-1 linked to PGIS. The FRET study found that the distance between COX-2-RFP and PGIS-CFP is shorter than that between COX-1-RFP and PGIS-CFP. SIGNIFICANCE: The study provided strong evidence suggesting that the low Km, faster ½t Vmax, and closer distance are the basis for COX-2 dominance over COX-1 (coupled to PGIS) in PGI2 synthesis, and further demonstrated the mechanisms of selective COX-2 inhibitors with higher potential to reduce synthesis of the vascular protector, PGI2.

A profile of NSAID-targeted arachidonic acid metabolisms in human embryonic stem cells (hESCs): implication of the negative effects of NSAIDs on heart tissue regeneration.

INTRODUCTION: An emerging technology using human embryonic stem cells (hESCs) to regenerate infarcted heart tissue has been underdeveloped. However, because non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, are taken during the infarction, it becomes critical to know whether the NSAIDs have negative impacts on heart tissue regeneration when using hESCs. METHODS: Mass spectrometry (LC/MS/MS) and high performance liquid chromatography (HPLC) analyses were used to analyze the functional presence of the elaborate prostanoids' biosynthesis and signaling systems in hESCs. The detected endogenous arachidonic acid (AA) released in the hESC membranes reflects the activity of phospholipase which directly controls the biosyntheses of the prostanoids. RESULTS: The complete inhibition of the endogenous prostaglandin E(2) (PGE(2)) biosynthesis by the cyclooxygenase-2 (COX-2) inhibitor, NS398, confirmed that the major prostanoids synthesized in the hESCs are mediated by the COX-2 enzyme. We also found that PGE(2) and the prostacyclin (PGI(2)) metabolite, 6-keto-PGF(1α), are present in the undifferentiated hESCs. CONCLUSION: This indicated different cyclooxygenase (COX)-downstream synthases and metabolizing enzymes are involved in the AA products' signaling through the COX-1 and COX-2 pathways. The presence of many enzymes' and receptors' [(COX-1, COX-2, microsomal prostaglandin E synthase (mPGES), cytosolic prostaglandin E synthase (cPGES), prostaglandin I synthase (PGIS), the PGE(2) subtype receptors (EP(1), EP(2), and EP(4)) and the prostacyclin receptor (IP)] involvement in the prostanoid biosynthesis and activity was confirmed by western blot. The studies implied the negative effects of NSAIDs, such as aspirin and COX-2 inhibitors, which suppress prostanoid production during tissue regeneration for infarcted heart when using hESCs.

Prostacyclin therapy for pulmonary arterial hypertension.

In pulmonary arterial hypertension, the blood vessels that carry blood between the heart and lungs are constricted, making it difficult for the heart to pump blood through the lungs. Prostacyclin, a prostanoid metabolized from endogenous arachidonic acid through the cyclooxygenase (COX) pathway, is a potent vasodilator that has been identified as one of the most effective drugs for the treatment of pulmonary arterial hypertension. Currently, prostacyclin and its analogues are widely used in the clinical management of pulmonary arterial hypertension patients. However, the mortality rate associated with pulmonary arterial hypertension has not been significantly reduced within the past 5 years. More powerful therapeutic approaches are needed. This article briefly reviews the current management of pulmonary arterial hypertension to identify the problems associated with present therapies; then it focuses on the emerging technology of prostacyclin synthase gene therapy and cell-based therapy using native stem cells and engineered stem cells with enhanced prostacyclin production capacity. By using the recent advances in technology and the molecular understanding of prostacyclin synthesis, researchers are prepared to make significant advances in the treatment of pulmonary arterial hypertension.

Instantly Converting Atrial Fibrillation into Sinus Rhythm by a Digital Rectal Exam on a 29-year-Old Male.

Vagal maneuvers cause increase in vagal tone, which has been shown to slow many types supraventricular tachycardia, such as atrial fibrillation (AF). However, the conversion of AF to sinus rhythm is usually not associated with vagal manuvers. Thus, AF is classically treated with medication and electrical cardioversion. Here, we present a 29-year-old male with no cardiovascular history and a low atherosclerotic risk profile who developed AF which converted into sinus rhythm immediately after a digital rectal exam. The patient remained asymptomatic after a 3-month follow-up. This implies that the digital rectal exam can be considered as an additional attempt to convert AF to sinus rhythm in AF patients.

Prunella stica inhibits the proliferation but not the prostaglandin production of Ishikawa cells.

Chinese herbs have been used to relieve dysmenorrhea associated with endometriosis. Active components in the herbs and their mechanisms of action remain unknown. Prunella stica, a Chinese herb commonly used to treat dysmenorrhea, was chosen for the present studies. Its effects were investigated on Ishikawa cells, an epithelial cell line derived from human endometrium. Cell proliferation and inhibition of interleukin 1beta (IL-1beta) induced prostaglandin (PG) production were examined. To learn more about the active components, 120 fractions were collected from the crude extract and each fraction was tested individually. To further characterize the active components, aliquots of fractions with activity were subject to mass spectrometry analysis. Crude extract of P. stica inhibited the proliferation of Ishikawa cells but not the IL-1beta induced PG production. Active components of P. stica clustered around fractions 64 and 92; they increased cell doubling time from 18.6 to 26.2 and 29.4h, respectively. Mass spectrometry analysis showed fractions 64 and 92 consisted of three components whose molecular weights were 337, 348 and 430 Daltons. The therapeutic effects of P. stica reside, in part, in inhibiting the proliferation of the epithelial cells derived from human endometrium. The active components are small molecules.

A strategy using NMR peptide structures of thromboxane A2 receptor as templates to construct ligand-recognition pocket of prostacyclin receptor.

BACKGROUND: Prostacyclin receptor (IP) and thromboxane A2 receptor (TP) belong to rhodopsin-type G protein-coupling receptors and respectively bind to prostacyclin and thromboxane A2 derived from arachidonic acid. Recently, we have determined the extracellular loop (eLP) structures of the human TP receptor by 2-D 1H NMR spectroscopy using constrained peptides mimicking the individual eLP segments. The studies have identified the segment along with several residues in the eLP domains important to ligand recognition, as well as proposed a ligand recognition pocket for the TP receptor. RESULTS: The IP receptor shares a similar primary structure in the eLPs with those of the TP receptor. Forty percent residues in the second eLPs of the receptors are identical, which is the major region involved in forming the ligand recognition pocket in the TP receptor. Based on the high homology score, the eLP domains of the IP receptor were constructed by the homology modeling approach using the NMR structures of the TP eLPs as templates, and then configured to the seven transmembrane (TM) domains model constructed using the crystal structure of the bovine rhodopsin as a template. A NMR structure of iloprost was docked into the modeled IP ligand recognition pocket. After dynamic studies, the segments and residues involved in the IP ligand recognition were proposed. A key residue, Arg173 involved in the ligand recognition for the IP receptor, as predicted from the modeling, was confirmed by site-directed mutagenesis. CONCLUSION: A 3-D model of the human IP receptor was constructed by homology modeling using the crystal structure of bovine rhodopsin TM domains and the NMR structures of the synthetic constrained peptides of the eLP domains of the TP receptor as templates. This strategy can be applied to molecular modeling and the prediction of ligand recognition pockets for other prostanoid receptors.

NMR structure of the thromboxane A2 receptor ligand recognition pocket.

To overcome the difficulty of characterizing the structures of the extracellular loops (eLPs) of G protein-coupled receptors (GPCRs) other than rhodopsin, we have explored a strategy to generate a three-dimensional structural model for a GPCR, the thromboxane A(2) receptor. This three-dimensional structure was completed by the assembly of the NMR structures of the computation-guided constrained peptides that mimicked the extracellular loops and connected to the conserved seven transmembrane domains. The NMR structure-based model reveals the structural features of the eLPs, in which the second extracellular loop (eLP(2)) and the disulfide bond between the first extracellular loop (eLP(1)) and eLP(2) play a major role in forming the ligand recognition pocket. The eLP(2) conformation is dynamic and regulated by the oxidation and reduction of the disulfide bond, which affects ligand docking in the initial recognition. The reduced form of the thromboxane A(2) receptor experienced a decrease in ligand binding activity due to the rearrangement of the eLP(2) conformation. The ligand-bound receptor was, however, resistant to the reduction inactivation because the ligand covered the disulfide bond and stabilized the eLP(2) conformation. This molecular mechanism of ligand recognition is the first that may be applied to other prostanoid receptors and other GPCRs.