Influencing Factors of HLA-DR Expression in Orbital Preadipocytes of Patients With Thyroid Associated Ophthalmopathy

Purpose: To investigate the effects of IFN-γ, IL-6, PPAR-γ agonist pioglitazone and PPAR-γ antagonist GW9662 on preadipocyte proliferation and HLA-DR expression.Methods: Preadipocytes were cultured from thyroid associated ophthalmopathy patients and health ocular trauma patients and induced to differentiate in vitro. IFN-γ, IL-6, PPAR-γ agonist pioglitazone and PPAR-γ antagonist GW9662 were added to preadipocytes to observe their effects on proliferation and HLA-DR expression.Results: IFN-γ significantly increased the levels of HLA-DR mRNA and protein in preadipocytes which are cultured from thyroid associated ophthalmopathy patients. IFN-γ also reduced the accumulation of lipid in the cytoplasm of preadipocytes. On the contrary, IL-6 had no effect on the expression of HLA-DR. Pioglitazone increased the expression of HLA-DR in orbital preadipocytes of TAO patients, and promoted the proliferation and intracellular lipid accumulation of preadipocytes. On the contrary, GW9662 decreased the expression of HLA-DR in orbital preadipocytes of TAO patients, and reduced the proliferation and intracellular lipid accumulation of preadipocytes.Conclusion: IFN-γ increase the expression of HLA-DR in TAO orbital preadipocytes after differentiation. PPAR - γ antagonists may be beneficial to the treatment of TAO.

Amorfrutins Relieve Neuropathic Pain through the PPARγ/CCL2 Axis in CCI Rats

Neuropathic pain is a public health problem. Although many pharmaceuticals are used to treat neuropathic pain, effective and safe drugs do not yet exist. In this study, we tested nociceptive responses in CCI rats, and ELISA assay was performed to examine the expression of proinflammatory cytokines. We found that amorfrutins significantly reduce the pain behaviors in CCI rats and suppress the expression of proinflammatory cytokines (TNFα, IL-6, and IL-1β) and chemokines (CCL2/CCR2) in the spinal cord. However, concurrent administration of a PPARγ antagonist, GW9662, reversed the antihyperalgesic effect induced by amorfrutins. The results indicate that amorfrutins inhibit the inflammation and chemokine expression by activating PPARγ, thus relieving neuropathic pain in CCI rats. Therefore, PPARγ-CCL2/CCR2 pathway might represent a new treatment option for neuropathic pain.

P720Atheroprotective effects of 17beta-estradiol are mediated by PPARgamma in human coronary artery smooth muscle cells

Background 17β-estradiol (E2) mediates vasculoprotection in various preclinical and clinical models of atherosclerosis and neointimal hyperplasia. However, the molecular mechanisms underlying these effects are still not fully elucidated. Previous studies have demonstrated the essential role of the peroxisome-proliferator-activated-receptor-γ (PPARγ) in mediating vasculoprotective effects of E2 in vivo. The aim of the current study was to investigate whether PPARγ is implicated in mediating vasculoprotective mechanisms of E2 in human coronary artery smooth muscle cells (HCASMC). Methods Primary HCASMC were purchased and stimulated with E2 [10 nM], the selective estrogen receptor α (ERα) agonist propylpyrazole triol (PPT) [50 nM] and the selective ERα antagonist methyl-piperidino-pyrazole (MPP) [1 μM], respectively. Changes in PPARγ mRNA and protein expression upon stimulation of ERα were assessed by qPCR and Western blot analyses. Nuclear PPARγ protein expression and DNA binding affinity was assessed after the isolation of the nuclear protein fraction. Hereafter, HCASMC were incubated with E2, PPARγ-antagonist GW9662 [1 μM – 30 μM], or both. HCASMC proliferation was assessed by nuclear BrdU staining and reactive oxygen species (ROS) formation was assessed by L-012- and DCF-DA assays. Results E2 significantly increased PPARγ expression in HCASMC (1.95±0.41 –fold; n=5; p=0.0335). This effect was mimicked by ERα agonist PPT (1.63±0.27 –fold; n=7; p=0.0489) and was abrogated by co-incubation with ERα antagonist MPP (1.17±0.18 –fold; n=3; pvs. control >0.05). Nuclear PPARγ expression was enhanced by E2 (1.53±0.16 –fold; n=4; pvs. control = 0.0074; Fig. 2A) whereas PPARγ's DNA binding activity to PPRE remained unchanged upon stimulation with E2 (0.94±0.11 –fold; n=4; pvs. control >0.05). Pharmacological inhibition of PI3K/Akt by LY294002 abrogated E2-induced expression of PPARγ (0.24±0.09 –fold; n=3; pvs. E2 = 0.0017), arguing for a PI3K/Akt-dependent activation by E2. The role of PPARγ in mediating vasculoprotective effects of E2 was assessed in functional assays using PPARγ-antagonist GW9662. E2 diminished HCASMC proliferation which was restored by GW9662. While E2 only slightly decreased ROS production by HCASMC, GW9662 significantly increased ROS levels (1,036±169 RLU x s–1 x cell–1 versus 561±99 RLU x s–1 x cell–1; n=5–6; p=0.0287). Conclusion In summary, the present study identifies PPARγ as a downstream mediator of E2-related atheroprotective effects in HCASMC. 17β-estradiol regulates vascular PPARγ-expression in HCASMC via the ERα receptor and the PI3K/Akt pathway. PPARγ agonism might be a promising therapeutic strategy to prevent cardiovascular events in postmenopausal women with depleted E2 plasma levels. Acknowledgement/Funding This work was supported by the Bonfor program of the University of Bonn [grant number O-109.0057 to JJ].

Abstract 461: Macrophage 12/15-lipoxygenase Controls the Development of Experimental Hypertension through PPAR?

Targeted disruption of the 12/15-lipoxygenase (Alox15) gene in mice prevents the development of angiotensin II-, DOCA-salt-, and L-NAME-induced experimental hypertension. Macrophages are the primary source of Alox15. Using Alox15-/- and wild type (WT) mice, we previously demonstrated that macrophage Alox15 is essential for experimental hypertension; however, the underlying mechanism is not known. Since Alox15-derived metabolites of arachidonic and linoleic acids are potent PPARγ agonists, we hypothesized that activation of macrophage PPARγ is the key step in Alox15 mediation of L-NAME-induced hypertension. Thioglycollate (TG) injection, used to elicit peritoneal macrophages, upregulated PPARγ protein expression and expression of its target gene CD36 in peritoneal macrophages, but not in aorta or kidney, of both WT (6.8±0.3 and 9.3±0.5 fold increase for PPARγ and CD36, respectively) and Alox15-/- mice (4.7±0.4 and 11.1±0.6 fold increase for PPARγ and CD36, respectively). Thus, TG bypassed Alox15 to activate PPARγ selectively in macrophages. Upregulation of PPARγ by TG restored the sensitivity to L-NAME hypertension (100 mg/kg/day L-NAME for 7 days) in Alox15-/- mice (129.4±7.3 mmHg for TG vs. 94.8±5.6 mmHg for non-injected controls, p<0.005). A similar hypertensive effect to L-NAME was observed with the adoptive transfer of TG-elicited Alox15-/- macrophages into Alox15-/- recipient mice (122.4±5.5 mmHg for macrophage vs. 96.2±6.2 mmHg for vehicle, p<0.05). The role of PPARγ was further specified by using the selective PPARγ antagonist, GW9662. When WT mice were treated with GW9662, 50 μg/kg/day for 12 days, L-NAME-induced hypertension did not develop (97.6±4.8 mmHg for GW9662 vs. 131.7±6.5 mmHg for vehicle, p<0.01). The PPARγ antagonist also prevented L-NAME-induced hypertension in TG-injected Alox15-/- mice, indicating a PPARγ-mediated effect in macrophages that results in hypertension. These results demonstrate that TG selectively upregulates macrophage PPARγ, bypassing the missing component in Alox15-/- macrophages, and permitting L-NAME-induced hypertension in Alox15-/- mice. Thus, the macrophage Alox15/PPARγ pathway is an important checkpoint in the development of L-NAME-induced hypertension.

Synergism between cAMP and PPARγSignalling in the Initiation of UCP1 Gene Expression in HIB1B Brown Adipocytes

Expression of the brown adipocyte-specific gene, uncoupling protein 1 (UCP1), is increased by both PPARγstimulation and cAMP activation through their ability to stimulate the expression of the PPAR coactivator PGC1α. In HIB1B brown preadipocytes, combination of the PPARγagonist, rosiglitazone, and the cAMP stimulator forskolin synergistically increased UCP1 mRNA expression, but PGC1αexpression was only increased additively by the two drugs. The PPARγantagonist, GW9662, and the PKA inhibitor, H89, both inhibited UCP1 expression stimulated by rosiglitazone and forskolin but PGC1αexpression was not altered to the same extent. Reporter studies demonstrated that combined rosiglitazone and forskolin synergistically activated transcription from a full length 3.1 kbp UCP1 luciferase promoter construct, but the response was only additive and much reduced when a minimal 260 bp proximal UCP1 promoter was examined. Rosiglitazone and forskolin in combination were able to synergistically stimulate promoters comprising of tandem repeats of either PPREs or CREs. We conclude that rosiglitazone and forskolin act together to synergistically activate the UCP1 promoter directly rather than by increasing PGC1αexpression and by a mechanism involving cross-talk between the signalling systems regulating the CRE and PPRE on the promoters.