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

Qing-Lan Ling,Emma Murdoch,Anita J Mohite,Shui-Ping So,Hironari Akasaka,Qun-Ying Li,Ke-He Ruan

doi:10.1038/s41598-018-19661-y

Qing-Lan Ling, Emma Murdoch + Show 5 more

Open Access

https://doi.org/10.1038/s41598-018-19661-y

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Abstract

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.

Highlights

Cellular prostanoid biosynthesis is regulated by cyclooxygenase (COX) enzymes, which exist in two isoforms (COX-1 and COX-2), and their downstream synthases
We first demonstrated that the microenvironment of cellular prostanoid biosynthesis in primary cells could be controlled by our Single-chain Enzyme Complex (SCHEC), COX-1-10aa-PGI2 synthase (PGIS) (Fig. 1ia)
Arachidonic acid metabolism was redirected in favor of prostaglandin I2 (PGI2) biosynthesis and disfavor to inflammatory PGE2 and thrombotic thromboxane A2 (TXA2) biosynthesis for vascular protection, which was demonstrated in vitro and in vivo in our previous publication[15]

Summary

Introduction

Cellular prostanoid biosynthesis is regulated by cyclooxygenase (COX) enzymes, which exist in two isoforms (COX-1 and COX-2), and their downstream synthases. The varied cells, including COS-7, HEK29311,12, primary cultured adipose stromal cells[13] and endothelial progenitor cells[14], were transfected with the single cDNA of COX-1-10aa-PGIS or COX-2-10aa-PGIS They showed high expression levels of the active hybrid enzyme with triple catalytic activities (converting AA into PGG2, PGH2 and into active PGI2), which resulted in redirecting the conversion of endogenous AA metabolites more toward the favorable PGI2 production rather than the unfavorable TXA2 and PGE2 production[11,12,13]. It greatly increased the possibility of correcting inherited heart diseases using emerging gene medicine, such as the transgene, COX-1-10aa-PGIS, at the current experimental level

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