ANTI-INFLAMMATORY AND ANTI-ATHEROGENIC EFFECTS OF 9E,11E-CONJUGATED LINOLEIC ACID

Open Access
- Author:
- Lee, Yunkyoung
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 04, 2008
- Committee Members:
- John Patrick Vanden Heuvel, Committee Chair/Co-Chair
Peter J Gillies, Committee Member
Pamela Hankey Giblin, Committee Member
Penny Margaret Kris Etherton, Committee Member
Andrea Marie Mastro, Committee Member
Peter John Hudson, Committee Member - Keywords:
- mTOR
anti-inflammatory
anti-atherogenic
CLA
PI3K - Abstract:
- Conjugated linoleic acid (CLA) is a mixture of dietary fatty acids that has various beneficial effects including a reduction of cancer, atherosclerosis and inflammation in animal models. Controversy exists on the specific isomers of CLA that are responsible for the benefits observed. CLA studies are predominantly performed with either a CLA mixture or with pure isomers, 9Z,11E- or 10E,12Z-CLA, due to their abundance in dairy products and dietary CLA supplements. These studies were conducted to examine how a specific CLA isomer, 9E,11E-CLA, regulates gene expression and markers of inflammation in a mouse macrophage cell line, RAW 264.7 (RAW). Microarray gene expression analysis of RAW cells treated with five different CLA isomers, 9E,11E-, 9Z,11E-, 9Z,11Z-, 10E,12Z- and 11Z,13E-CLA, revealed distinct biological functions and properties of the five CLA isomers. Among the isomers tested, 9E,11E-, 9Z,11Z-, 10E,12Z-, and 11Z,13E-CLA decreased LPS-induced pro-inflammatory cytokine mRNA levels, IL-1alpha, IL-1beta and IL-6. 9E,11E-CLA was unique in that it produced a robust induction of interleukin 1 receptor antagonist (IL-1Ra) without altering other pro-inflammatory cytokines. Although 9E,11E-CLA is a weak ligand for peroxisome proliferator-activated receptors (PPARs), it was not able to explain the uniqueness of 9E,11E-CLA regulating IL-1Ra. Coactivator recruitment by CLA isomers showed their distinct properties as selective receptor modulators for PPARgamma and RXRalpha. IL-1Ra, an endogenous antagonist of IL-1 signaling, is induced by various factors. We hypothesized that the anti-inflammatory effects of 9E,11E-CLA are mediated via the induction of IL-1Ra in RAW cells; consequently, influencing atherogeneic-related gene regulation in human umbilical vein endothelial cells (HUVEC) which is a commonly used in vitro model for endothelial biology. IL-1Ra protein levels were decreased in RAW cells by siRNA to determine the importance of IL-1Ra in the anti-inflammatory effects of 9E,11E-CLA. Blocking IL-1Ra abrogated the capability of 9E,11E-CLA to decrease LPS-induced IL-1alpha and IL-6 mRNA levels, but IL-1beta was not affected which suggests the importance of IL-1Ra in regulating the anti-inflammatory property via 9E,11E-CLA. A response element for 9E,11E-CLA in the IL-1Ra promoter sequence was not detected, but we identified that the IL-1Ra induction by 9E,11E-CLA is different than the LPS induction of IL-1Ra. Blocking the PI3K pathway via LY294002 and the mTOR pathway via rapamycin abolished the 9E,11E-CLA induced-IL-1Ra, which indicates the PI3K/mTOR pathway is responsible for the induction of IL-1Ra by 9E,11E-CLA. Since atherogenesis is an inflammatory disease, anti-inflammatory agents can be beneficial to decrease atherogenic-related genes; therefore, potential anti-atherogenic effects of 9E,11E-CLA were measured in HUVEC. We observed that 9E,11E-CLA decreased atherogenic-related genes such as ICAM-1, VCAM-1, MCP-1, E-selectin, P-selectin, and CCR-2 in HUVEC. The alteration of the adhesion molecules by 9E,11E-CLA results in a decrease of cell-to-cell interaction; however, recombinant IL-1Ra did not alter the adhesion activity of RAW and HUVEC. HUVEC treated with siIL-1Ra RAW-conditioned media induced significantly higher levels of ICAM-1, VCAM-1, MCP-1, and E-selectin than cells treated with control media. These results suggests that factors secreted from siIL-1Ra RAW cells enhance the adhesion molecules, and 9E,11E-CLA's anti-atherogenic effect may be secondary to IL-1Ra level alteration. These studies revealed not only 9E,11E-CLA itself, but also factors secreted from siIL-1Ra RAW cells affect the atherogeneic-related factors in HUVEC. Taken together, 9E,11E-CLA has anti-inflammatory effects and potential anti-atherogenic properties in in vitro models which may suggest a therapeutic use in the in vivo models.