Creating a mouse model resistant to induced ischemic stroke and cardiovascular damage.

Vascular prostanoids, isomerized from an intermediate prostaglandin (PG), H2, produced by cyclooxygenase (COX), exert various effects on the vascular system, both protective and destructive. During endothelial dysfunction, vascular protector prostacyclin/prostaglandin I2 (PGI2) is decreased, while inflammatory PGE2 and thrombotic TXA2 are increased. Therefore, our research aim was to reverse the event by controlling PGH2 metabolism by generating an in vivo model via enzymatic engineering of COX-1 and prostacyclin synthase (PGIS). The COX-1 and PGIS genes were linked to a 10-residue amino acid linker to form a Single-chain Enzyme Complex (SCHEC), COX-1-10aa-PGIS. Transgenic (CP-Tg) mice in a FVB/N background were generated using the pronuclear microinjection method. We first confirmed mRNA and protein expression of COX-1-10aa-PGIS in various CP-Tg mouse tissues, as well as upregulation of circulating PGI2. We then examined the cardiovascular function of these mice. Our CP-Tg mice exhibited marked resistance to vascular assault through induced carotid arterial blockage, acute thrombotic stroke and arterial arrest, angiotensin-induced peripheral vasoconstriction, and hepatic lipid accumulation after receiving a high-fat diet. They also had a longer lifespan compared with wild-type mice. This study raises the possibility of fighting cardiovascular diseases by regulating cellular arachidonic acid-derived PGH2 metabolites using enzymatic engineering.

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.

Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells.

Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2-min) half-life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1 (COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat bone marrow mononuclear cells. PGI2-secreting strains (PGI2-EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT-PCR, and Western blot analyses confirmed the overexpression of COX-1-10aa-PGIS in PGI2-EPCs. PGI2-EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (P < 0.05) and showed higher intrinsic angiogenic capability; conditioned medium (CM) from PGI2-EPCs promoted better tube formation than CM from native EPCs (P < 0.05). Cell- and paracrine-mediated in vitro angiogenesis was attenuated when COX-1-10aa-PGIS protein expression was knocked down. Whole-cell patch-clamp studies showed that 4-aminopyridine-sensitive K(+) current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2-EPCs (7.50 ± 1.59 pA/pF; P < 0.05) compared with rSMCs cocultured with native EPCs (3.99 ± 1.26 pA/pF). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2-EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine-mediated cellular protections, including promoting in vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia.

Novel mechanism of the vascular protector prostacyclin: regulating microRNA expression.

Prostacyclin (PGI(2)) is a key vascular protector, metabolized from endogenous arachidonic acid (AA). Its actions are mediated through the PGI(2) receptor (IP) and nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ). Here, we found that PGI(2) is involved in regulating cellular microRNA (miRNA) expression through its receptors in a mouse adipose tissue-derived primary culture cell line expressing a novel hybrid enzyme gene (COX-1-10aa-PGIS), cyclooxygenase-1 (COX-1) and PGI(2) synthase (PGIS) linked with a 10-amino acid linker. The triple catalytic functions of the hybrid enzyme in these cells successfully redirected the endogenous AA metabolism toward a stable and dominant production of PGI(2). The miRNA microarray analysis of the cell line with upregulated PGI(2) revealed a significant upregulation (711, 148b, and 744) and downregulation of miRNAs of interest, which were reversed by antagonists of the IP and PPARγ receptors. Furthermore, we also found that the insulin-mediated lipid deposition was inhibited in the PGI(2)-upregulated adipocytes. The study also initiated a discussion that suggested that the endogenous PGI(2) inhibition of lipid deposition in adipocytes could involve miRNA-mediated inhibition of expression of the targeted genes. This indicated that PGI(2)-miRNA regulation could exist in broad pathophysiological processes involving PGI(2) (i.e., apoptosis, vascular inflammation, cancer, embryo implantation, and obesity).