Date of Award

12-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular Therapeutics and Cell Signaling

Research Advisor

Gabor J. Tigyi, M.D., Ph.D.

Committee

Polly A. Hofmann, Ph.D. Edwards A. Park, Ph.D. Abby L. Parrill, Ph.D. Gadiparthi N. Rao, Ph.D.

Keywords

lysophosphatidic acid, cyclic phosphatidic acid, phospholipase D, neointima, atherosclerosis, PPAR!

Abstract

Lysophosphatidic acid (LPA) and its ether analog alkyl glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor γ (PPARγ). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARγ agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for 1 hour without injury into the carotid arteries of LPA1, LPA2, LPA1&2 double knockout, and Mx1Cre-inducible conditional PPARγ knockout mice targeted to vascular smooth muscle cells, macrophages, and endothelial cells. The neointima was quantified and also stained for CD31, CD68, CD11b, and "-smooth muscle actin markers. In LPA1, LPA2, LPA1&2 GPCR knockouts, Mx1Cre transgenic, PPARγ fl/- , and uninduced Mx1Cre#PPAR! fl/- mice AGP- and ROSI-elicited neointima was indistinguishable in its progression and cytological features from that of WT C57BL/6 mice. In PPARγ -/- knockout mice, generated by activation of Mx1Cre-mediated recombination, AGP and ROSI failed to elicit neointima and vascular wall remodeling. Our findings point to a difference in the effects of AGP and ROSI between the balloon-injury- and the non-injury chemically-induced neointima. The present data provide genetic evidence for the requirement of PPARγ in AGP- and ROSI-elicited neointimal thickening in the non-injury model and reveal that the overwhelming majority of the cells in the neointimal layer express !-smooth muscle actin.

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature’s simplest phospholipids, is found in cells from slime mold to humans and has largely unknown function. We find that CPA is generated in mammalian cells in a stimulus coupled-manner by phospholipase D2 (PLD2), and binds to and inhibits the nuclear hormone receptor PPAR! with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPAR! target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in vivo. Inhibition of PLD2 by shRNA, a dominant negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARγ inhibition. We conclude that CPA is a novel second messenger and a physiological inhibitor of PPARγ, revealing that PPARγ is regulated by endogenous agonists as well as by antagonists.

PPAR" is a nuclear hormone receptor related to many human diseases, including obesity, atherosclerosis, diabetes, and cancers. Recent studies have provided evidence that LPA and its analog AGP activate PPARγ. On the other hand, CPA, similar in structure to LPA, can be generated by PLD2 and negatively regulates PPARγ functions. PPARγ agonists elicit lipid accumulation in macrophages and arterial wall remodeling when topically applied to the carotid artery in the rat non-injury model. Stimulation of PLD2 protects the carotid artery from PPARγ-mediated neointima formation. Consistent with this, inhibition of PLD2 activity using a PLD inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide, diminishes the protective effect of insulin. Stimulation of PPARγ by AGP leads to the recruitment of circulating vascular progenitor cells into the vessel wall and the cells of the ensuing neointima express the α-smooth muscle actin marker. We summarize our current knowledge about the mechanism of PPARγ elicits in response to the endogenous lysophosphatidic acid analogs LPA, AGP, CPA, and the methodological challenges that one faces when working on the intracellular action of LPA.

DOI

10.21007/etd.cghs.2011.0324

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