Endothelium-specific interference with peroxisome proliferator activated receptor gamma causes cerebral vascular dysfunction in response to a high-fat diet

Abstract

The ligand-activated transcription factor peroxisome proliferator activated receptor gamma (PPARgamma) is expressed in vascular endothelium where it exerts anti-inflammatory and antioxidant effects. However, its role in regulating vascular function remains undefined. We examined endothelial function in transgenic mice expressing dominant-negative mutants of PPARgamma under the control of an endothelial-specific promoter to test the hypothesis that endothelial PPARgamma plays a protective role in the vasculature. Under baseline conditions, responses to the endothelium-dependent agonist acetylcholine were not affected in either aorta or the basilar artery in vitro. In response to feeding a high-fat diet for 12 weeks, acetylcholine produced dilation that was markedly impaired in the basilar artery of mice expressing dominant-negative mutants, but not in mice expressing wild-type PPARgamma controlled by the same promoter. Unlike basilar artery, 12 weeks of a high-fat diet was not sufficient to cause endothelial dysfunction in the aorta of mice expressing dominant-negative PPARgamma, although aortic dysfunction became evident after 25 weeks. The responses to acetylcholine in basilar artery were restored to normal after treatment with a scavenger of superoxide. Baseline blood pressure was only slightly elevated in the transgenic mice, but the pressor response to angiotensin II was augmented. Thus, interference with PPARgamma in the endothelium produces endothelial dysfunction in the cerebral circulation through a mechanism involving oxidative stress. Consistent with its role as a fatty acid sensor, these findings provide genetic evidence that endothelial PPARgamma plays a critical role in protecting blood vessels in response to a high-fat diet.

Figure 1. Generation of E-PPARγ Transgenic Mice

A. A schematic representation of the transgenic construct. B. Southern blot analysis of E-WT, E-P467L and E-V290M lines used in this report. The position of the endogenous mouse PPARγ (mPPARγ) and transgene (hPPARγ) is indicated. The lines marked by * are those used in this report. There was no correlation between transgene copy number and expression.

Figure 2. Expression of PPARγ in the Blood Vessel Wall

Expression of endogenous mPPARγ with hPPARγ transgene in the lung and aorta assayed by RPA. The aorta RNA is actually a combination of aorta and carotid artery pooled from several transgenic mice of the indicated line and construct. Cyclophilin expression is the internal control. h, human; m, mouse.

Figure 3. Cell-Specific Expression of E-V290M Transgene

Cell-specificity of transgene expression was analyzed via RT-PCR. SMMHC and VeCad were used as markers for smooth muscle and endothelial cells, respectively. GAPDH was used as an internal control. PPARγ primers were specific for the hPPARγ transgene. The presence of RT and endothelium (E) is indicated by + and −.

Figure 4. Endothelial Function in Basilar Artery Under Normal Diet

Vascular function was measured in E-V290M (A–D) and E-P467L (E–H) mice compared with non-transgenic littermates. Relaxation was measured in response to Ach (A,E), A23187 (B,F), and papaverine (C,G) and contraction was measured in response to KCl (D,H).

Figure 5. Endothelial Function in E-V290M and E-P467L Basilar Artery After High Fat Diet

Vascular function was measured in E-V290M (A–D) and E-P467L (E–H) mice compared with non-transgenic littermates. Relaxation was measured in response to Ach (A,E), A23187 (B,F), and papaverine (C,G) and contraction was measured in response to KCl (D,H). *, P<0.05 vs non-transgenic.

Figure 6. Endothelial Function in E-WT Basilar Artery After High Fat Diet

Vascular function was measured in E-WT mice compared with non-transgenic littermates. Relaxation was measured in response to Ach (A), A23187 (B), and papaverine (C) and contraction was measured in response to KCl (D).

Figure 7. Oxidative Stress

A. Vascular function was measured in E-V290M mice ± high fat diet and Tempol as indicated. *, P<0.05 vs high fat diet plus Tempol. B. Expression of select genes in the oxidative stress pathway as measured by microarray analysis. Each bar reflects the change in gene expression in E-V290M vs NT as a composite of 3 arrays each from NT and E-V290M. In cases where multiple probe sets were available, they were averaged. *, P<0.05 after correction for multiple comparisons.

Figure 8. Blood Pressure of E-V290M Mice

A–B. Arterial blood pressure was measured via radiotelemetry in mice maintained on a normal diet during the day (A) and night (B). C. Systolic blood pressure was measured by tail cuff before (open) and after (closed) angiotensin-II infusion in mice fed a normal diet. *, P <0.01 vs. untreated. D. Increase in SBP after angiotensin-II infusion. *, P=0.013 vs. NT.