Endothelial Dysfunction Induced by Mitochondrial Uncoupling is prevented by Retinol Binding Protein 7, a PPARγ Target Gene

Abstract

PPARγ is a nuclear receptor that prevents oxidative stress in the endothelium. Endothelium specific PPARγ target gene, retinol binding protein 7 (RBP7) is required for the protective effects of PPARγ. In response to a cardiovascular stressor (high fat diet or Ang II), transgenic mice carrying a dominant‐negative mutation in PPARγ in the endothelium (E‐V290M) and RBP7 knockout mice (RBP7KO) exhibited increased oxidative stress and endothelial dysfunction. We hypothesize that RBP7 has a protective role against endothelial dysfunction induced by the pro‐oxidant effects of mitochondrial uncoupling (MU). Acetylcholine (ACh) and sodium nitroprusside (SNP) induced vasodilation response were evaluated in carotid arteries from 4 and 8 month old RBP7KO mice, infused with either saline or a sub‐pressor dose of Ang II (120ng/kg/min) for 2 weeks. Ang II infusion did not alter systolic blood pressure either in wild type (WT) (4 months old: 116.2±4.2 vs 113.9±4.1 mmHg; 8 months old: 111.3±3.8 vs 109.5±2.9 mmHg) or RBP7KO (4 months old: 112.9±1.1 vs 111.8±5.2 mmHg; 8 months old: 112.2±4.2 vs 113.9±4.1 mmHg). Endothelium‐dependent relaxation to ACh was not affected by Ang II in either RBP7KO (4 month old: % relaxation ‐ saline, 89.7±1.3 vs Ang II, 93.8±2.0; 8 months old: % relaxation ‐ saline, 80.1±6.8 vs Ang II, 74.6±10.6) or WT littermates (4 months old: % relaxation ‐ saline, 98.1±0.3 vs Ang II, 94.5±1.7; 8 months old: % relaxation ‐ saline, 81.4±4.7 vs Ang II, 78.6±10.4). To test if RBP7 plays a role in MU, the vessels were pre‐incubated with the MU carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; 1uM for 30 min). Pre‐incubation with CCCP did not alter the relaxation responses to ACh in 4 month old mice in all groups. However, in 8 month old mice, there is a trend towards impaired endothelial function in the carotid artery from Ang II‐infused RBP7KO compared with genotype‐matched saline‐treated mice (% relaxation: 51.9±3.8 vs 81.8±5.3, p=0.06) as well as Ang II‐treated WT mice (% relaxation: 51.9±3.8 vs 77.1±2.0 p=0.08). Giving that, RBP7 may play a role in MU, we analyzed the protein expression (western blot) of fusion (OPA1) and fission (DRP1) markers in RBP7 KO and WT mouse lung endothelial cells (MLEC) incubated with CCCP (10 uM) for 15, 30 and 60 minutes. At baseline, both, long (L) (3.62±0.19 vs 3.41±0.13 AU) and short (S) (1.01±0.07 vs 0.97±0.04) isoform of OPA1 were equally expressed in WT and RBP7 KO MLEC respectively. The expression of L OPA1 was almost inhibited in WT (0.12 ± 0.01 AU) and RBP7 KO (0.24 ± 0.01 AU) MLEC after 60 min, while there was no effect in S OPA1. We speculate that differences in L OPA 1 expression may be observed with a lower dose of CCCP or in a recovery experiment where the CCCP is replaced by new media for couple of hours. No differences were observed in neither, the phosphorylation or expression of DRP1.Although the role of RBP7 in fusion/fission process still unclear, we conclude that Ang II alone was not able to impair vascular function, however, in 8 month old mice, Ang II increased the susceptibility to CCCP‐dependent endothelial impairment, and RBP7 was required to mediate vascular protection against mitochondrial uncoupling.