Effect of Selective Up regulation of Prostacyclin Synthesis On Its Signaling And Vascular Protection



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Prostacyclin (PGI2) is a very important endogenous vascular protector. It provides vascular protection through actions like vasodilatation, platelet aggregation and vascular smooth muscle cell proliferation inhibition and smooth muscle differentiation regulation. Therefore since decades, there have been many ongoing attempts to use PGI2 to treat vascular disorders such as pulmonary arterial hypertension, peripheral artery disease and thrombosis. However, the short half-life of PGI2 has limited its therapeutic potential. PGI2 is synthesized endogenously from arachidonic acid using enzymes cyclooxygenase isoform 1/2(COX-1/2) and prostacyclin synthase (PGIS). Single gene therapy with either COX or PGIS through adenoviral vectors does not specifically overproduce PGI2 and also is associated with inflammatory reactions. Thus to address these problems, our lab engineered a single hybrid gene (COX-1-10aa-PGIS) by fusing COX-1 with PGIS through transmembrane linker of 10 amino acids (10aa). Adipose tissue derived cells have been used therapeutically in several vascular disorders due to their regenerative properties. Thus we isolated and cultured mature adipocytes (MA) from mouse adipose tissue and called it as prostanoid synthesizing fat cells (PSFC). Further, we engineered a novel adipose tissue-derived cell that constantly produces PGI2, through transfecting of the engineered cDNA of the hybrid enzyme (human COX-1-10-aa-PGIS), which has superior triple catalytic functions in directly converting arachidonic acid into PGI2. The gene-transfected cells were further converted into a stable cell line, in which the cells constantly expressed the hybrid enzyme and were capable of overproducing specifically PGI2 and thus we called this cell line as PGI2 producing PSFC (PGI2-PSFC). When the results of the HPLC activity assay and LC/MS/MS were compared between just vector transfected (PSFC) and COX-1-10aa-PGIS gene transfected cells (PGI2-PSFC), it was observed that the majority of the endogenous AA metabolism shifted from that of unwanted PGE2 (in PSFC) to that of the preferred PGI2 (in PGI2-PSFC) with a PGI2/PGE2 ratio change from 0.03 to 25. The PGI2-producing cell line not only exhibited an approximate 50-fold increase in PGI2 biosynthesis, but also demonstrated superior anti-platelet aggregation in vitro, and increased reperfusion in the mouse ischemic hindlimb and thrombogenesis models in vivo. PGI2 regulates functions like vascular smooth muscle cell differentiation and proliferation and apoptosis. These actions are mediated through the PGI2 receptor (IP) and nuclear receptor, peroxisome proliferator-activated receptors (PPAR). MicroRNAs (miRNAs), which are negative regulators of gene expression, also regulate similar functions like PGI2. PGI2 regulates expression of genes by signaling through PPARs. Thus to find out if PGI2 regulates gene expression by regulating miRNA expression by signaling through the IP and PPAR, a miRNA microarray analysis of the PGI2-PSFCs and PSFCs was performed. Analysis of the results obtained from miRNA study, revealed that PGI2 is involved in regulating cellular microRNA (miRNA) expression through its receptors in the mouse adipose tissue-derived primary culture cell line overexpressing COX-1-10aa-PGIS (PGI2-PSFC). The miRNA microarray analysis of the PGI2-PSFC revealed a significant up-regulation (711, 148b, and 744) and down-regulation of miRNAs of interest, which were reversed by antagonists to the IP and PPARγ receptors. Furthermore, we also found that the insulin-mediated lipid deposition was inhibited in the PGI2-PSFCs. The study also initiated a discussion, which suggested that the endogenous PGI2 inhibition of lipid deposition in adipocytes could involve miRNA-mediated inhibition of expression of the targeted genes specifically of Akt1. To translate the results of in vitro study, a transgenic mouse permanently expressing the COX-1-10aa-PGIS gene was generated. This genetic mouse animal model was used to study the effect of COX-1-10aa-PGIS overexpression on PGI2 production in vivo and as well as its vascular protective effect. The transgenic mice was genotyped using polymerase chain reaction and further bred to develop a colony of homozygous transgenic mice. Quantitative PCR was performed to distinguish between the hemizygous and homozygous mice. PCR and imunoblot analysis of all the organs and tissues of mice revealed the expression of COX-1-10aa-PGIS in adipose tissue, brain, heart and uterus. LC/MS analysis of urine and plasma revealed approximately five-folds increase in PGI2 biosynthesis permanently in vivo. The animals showed strong resistance to photochemical induced thrombosis. They produced healthy pups with a an increase in live births when compared to the wild type without significant adverse effects in development and functions of major organs including brain, heart, kidneys. Tail cuff method was used to measure blood pressure of the mice which was similar to that in the wild type. Thus PGI2-PSFCs (therapeutic cells) can be used as cell therapy as an alternative to adenoviral gene transfer to treat ischemic and vascular conditions. The cells, which have an ability to increase the biosynthesis of the vascular protector, PGI2, while reducing that of the vascular inflammatory mediator, PGE2, 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 PGI2-deficient diseases, such as pulmonary arterial hypertension. PGI2 mediated gene expression regulation through miRNA expression regulation has unraveled a novel signaling mechanism for PGI2. This signaling could exist in broad pathophysiological processes involving PGI2 (i.e. apoptosis, vascular inflammation, cancer, embryo implantation, and obesity). The results of the transgenic mice overexpressing COX-1-10aa-PGIS and producing PGI2 provides the first evidences that risk of stroke and heart diseases could be reduced by the human transgene, as well as the insight into the possible future transgenic medicine to prevent and reduce the genetic risk of heart disease.