Ko-Ting Lu

Interference with PPARγ in endothelium accelerates angiotensin II-induced endothelial dysfunction

The ligand activated nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in the endothelium regulates vascular function and blood pressure (BP). We previously reported that transgenic mice (E-V290M) with selectively targeted endothelial-specific expression of dominant negative PPARγ exhibited endothelial dysfunction when treated with a high-fat diet, and exhibited an augmented pressor response to angiotensin II (ANG II). We hypothesize that interference with endothelial PPARγ would exacerbate ANG II-induced endothelial dysfunction. Endothelial function was examined in E-V290M mice infused with a subpressor dose of ANG II (120 ng·kg(-1)·min(-1)) or saline for 2 wk. ANG II infusion significantly impaired the responses to the endothelium-dependent agonist acetylcholine both in basilar and carotid arteries from E-V290M but not NT mice. This impairment was not due to increased BP, which was not significantly different in ANG II-infused E-V290M compared with NT mice. Superoxide levels, and expression of the pro-oxidant Nox2 gene was elevated, whereas expression of the anti-oxidant genes Catalase and SOD3 decreased in carotid arteries from ANG II-infused E-V290M mice. Increased p65 and decreased Iκ-Bα suggesting increased NF-κB activity was also observed in aorta from ANG II-infused E-V290M mice. The responses to acetylcholine were significantly improved both in basilar and carotid arteries after treatment with Tempol (1 mmol/l), a scavenger of superoxide. These findings provide evidence that interference with endothelial PPARγ accelerates ANG II-mediated endothelial dysfunction both in cerebral and conduit arteries through an oxidative stress-dependent mechanism, suggesting a role for endothelial PPARγ in protecting against ANG II-induced endothelial dysfunction.

Protective Role for Tissue Inhibitor of Metalloproteinase-4, a Novel Peroxisome Proliferator–Activated Receptor-γ Target Gene, in Smooth Muscle in Deoxycorticosterone Acetate–Salt Hypertension

Loss of peroxisome proliferator–activated receptor-&ggr; (PPAR&ggr;) function causes hypertension, whereas its activation lowers blood pressure. Evidence suggests that these effects may be attributable to PPAR&ggr; activity in the vasculature. However, the specific transcriptional targets of PPAR&ggr; in vessels remain largely unidentified. In this study, we examined the role of smooth muscle PPAR&ggr; during salt-sensitive hypertension and investigated its transcriptional targets and functional effect. Transgenic mice expressing dominant-negative PPAR&ggr; (S-P467L) in smooth muscle cells were more prone to deoxycorticosterone acetate–salt–induced hypertension and mesenteric arterial dysfunction compared with nontransgenic controls. Despite similar morphometry at baseline, vascular remodeling in conduit and small arteries was enhanced in S-P467L after deoxycorticosterone acetate–salt treatment. Gene expression profiling in aorta and mesenteric arteries revealed significantly decreased expression of tissue inhibitor of metalloproteinase-4 (TIMP-4) in S-P467L. Expression of TIMP-4 was increased by deoxycorticosterone acetate–salt treatment, but this increase was ablated in S-P467L. Interference with PPAR&ggr; activity either by treatment with a PPAR&ggr; inhibitor, GW9662, or by expressing P467L PPAR&ggr; markedly suppressed TIMP-4 in primary smooth muscle cells. PPAR&ggr; binds to a PPAR response element (PPRE) in chromatin close to the TIMP-4 gene in smooth muscle cells, suggesting that TIMP-4 is a novel target of PPAR&ggr;. The interference with PPAR&ggr; and decrease in TIMP-4 were accompanied by an increase in total matrix metalloproteinase activity. PPAR&ggr;-mediated loss of TIMP-4 increased, whereas overexpression of TIMP-4 decreased smooth muscle cell migration in a scratch assay. Our findings highlight a protective mechanism induced by PPAR&ggr; in deoxycorticosterone acetate–salt treatment, establishing a novel mechanistic link between PPAR&ggr; and TIMP-4.

Effect of selective expression of dominant-negative PPARγ in pro-opiomelanocortin neurons on the control of energy balance

Peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of adipogenesis, was recently shown to affect energy homeostasis through its actions in the brain. Deletion of PPARγ in mouse brain, and specifically in the pro-opiomelanocortin (POMC) neurons, results in resistance to diet-induced obesity. To study the mechanisms by which PPARγ in POMC neurons controls energy balance, we constructed a Cre-recombinase-dependent conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ and the tdTomato reporter. Inducible expression of both forms of PPARγ was validated in cells in culture, in liver of mice infected with an adenovirus expressing Cre-recombinase (AdCre), and in the brain of mice expressing Cre-recombinase either in all neurons (NES(Cre)/PPARγ-P467L) or selectively in POMC neurons (POMC(Cre)/PPARγ-P467L). Whereas POMC(Cre)/PPARγ-P467L mice exhibited a normal pattern of weight gain when fed 60% high-fat diet, they exhibited increased weight gain and fat mass accumulation in response to a 10% fat isocaloric-matched control diet. POMC(Cre)/PPARγ-P467L mice were leptin sensitive on control diet but became leptin resistant when fed 60% high-fat diet. There was no difference in body weight between POMC(Cre)/PPARγ-WT mice and controls in response to 60% high-fat diet. However, POMC(Cre)/PPARγ-WT, but not POMC(Cre)/PPARγ-P467L, mice increased body weight in response to rosiglitazone, a PPARγ agonist. These observations support the concept that alterations in PPARγ-driven mechanisms in POMC neurons can play a role in the regulation of metabolic homeostasis under certain dietary conditions.

Retinol-binding protein 7 is an endothelium-specific PPARγ cofactor mediating an antioxidant response through adiponectin

Impaired PPARγ activity in endothelial cells causes oxidative stress and endothelial dysfunction which causes a predisposition to hypertension, but the identity of key PPARγ target genes that protect the endothelium remain unclear. Retinol-binding protein 7 (RBP7) is a PPARγ target gene that is essentially endothelium specific. Whereas RBP7-deficient mice exhibit normal endothelial function at baseline, they exhibit severe endothelial dysfunction in response to cardiovascular stressors, including high-fat diet and subpressor angiotensin II. Endothelial dysfunction was not due to differences in weight gain, impaired glucose homeostasis, or hepatosteatosis, but occurred through an oxidative stress-dependent mechanism which can be rescued by scavengers of superoxide. RNA sequencing revealed that RBP7 was required to mediate induction of a subset of PPARγ target genes by rosiglitazone in the endothelium including adiponectin. Adiponectin was selectively induced in the endothelium of control mice by high-fat diet and rosiglitazone, whereas RBP7 deficiency abolished this induction. Adiponectin inhibition caused endothelial dysfunction in control vessels, whereas adiponectin treatment of RBP7-deficient vessels improved endothelium-dependent relaxation and reduced oxidative stress. We conclude that RBP7 is required to mediate the protective effects of PPARγ in the endothelium through adiponectin, and RBP7 is an endothelium-specific PPARγ target and regulator of PPARγ activity.

Hypertension-Causing Mutation in Peroxisome Proliferator–Activated Receptor γ Impairs Nuclear Export of Nuclear Factor-κB p65 in Vascular Smooth Muscle

Selective expression of dominant negative (DN) peroxisome proliferator–activated receptor &ggr; (PPAR&ggr;) in vascular smooth muscle cells (SMC) results in hypertension, atherosclerosis, and increased nuclear factor-&kgr;B (NF-&kgr;B) target gene expression. Mesenteric SMC were cultured from mice designed to conditionally express wild-type (WT) or DN-PPAR&ggr; in response to Cre recombinase to determine how SMC PPAR&ggr; regulates expression of NF-&kgr;B target inflammatory genes. SMC-specific overexpression of WT-PPAR&ggr; or agonist-induced activation of endogenous PPAR&ggr; blunted tumor necrosis factor &agr; (TNF-&agr;)–induced NF-&kgr;B target gene expression and activity of an NF-&kgr;B–responsive promoter. TNF-&agr;–induced gene expression responses were enhanced by DN-PPAR&ggr; in SMC. Although expression of NF-&kgr;B p65 was unchanged, nuclear export of p65 was accelerated by WT-PPAR&ggr; and prevented by DN-PPAR&ggr; in SMC. Leptomycin B, a nuclear export inhibitor, blocked p65 nuclear export and inhibited the anti-inflammatory action of PPAR&ggr;. Consistent with a role in facilitating p65 nuclear export, WT-PPAR&ggr; coimmunoprecipitated with p65, and WT-PPAR&ggr; was also exported from the nucleus after TNF-&agr; treatment. Conversely, DN-PPAR&ggr; does not bind to p65 and was retained in the nucleus after TNF-&agr; treatment. Transgenic mice expressing WT-PPAR&ggr; or DN-PPAR&ggr; specifically in SMC (S-WT or S-DN) were bred with mice expressing luciferase controlled by an NF-&kgr;B–responsive promoter to assess effects on NF-&kgr;B activity in whole tissue. TNF-&agr;–induced NF-&kgr;B activity was decreased in aorta and carotid artery from S-WT but was increased in vessels from S-DN mice. We conclude that SMC PPAR&ggr; blunts expression of proinflammatory genes by inhibition of NF-&kgr;B activity through a mechanism promoting nuclear export of p65, which is abolished by DN mutation in PPAR&ggr;.

Estrogen Receptor α Is Required for Maintaining Baseline Renin Expression

Enzymatic cleavage of angiotensinogen by renin represents the critical rate-limiting step in the production of angiotensin II, but the mechanisms regulating the initial expression of the renin gene remain incomplete. The purpose of this study is to unravel the molecular mechanism controlling renin expression. We identified a subset of nuclear receptors that exhibited an expression pattern similar to renin by reanalyzing a publicly available microarray data set. Expression of some of these nuclear receptors was similarly regulated as renin in response to physiological cues, which are known to regulate renin. Among these, only estrogen receptor &agr; (ER&agr;) and hepatic nuclear factor &agr; have no known function in regulating renin expression. We determined that ER&agr; is essential for the maintenance of renin expression by transfection of small interfering RNAs targeting Esr1, the gene encoding ER&agr;, in renin-expressing As4.1 cells. We also observed that previously characterized negative regulators of renin expression, Nr2f2 and vitamin D receptor, exhibited elevated expression in response to ER&agr; inhibition. Therefore, we tested whether ER&agr; regulates renin expression through an interaction with Nr2f2 and vitamin D receptor. Renin expression did not return to baseline when we concurrently suppressed both Esr1 and Nr2f2 or Esr1 and vitamin D receptor mRNAs, strongly suggesting that Esr1 regulates renin expression independent of Nr2f2 and vitamin D receptor. ER&agr; directly binds to the hormone response element within the renin enhancer region. We conclude that ER&agr; is a previously unknown regulator of renin that directly binds to the renin enhancer hormone response element sequence and is critical in maintaining renin expression in renin-expressing As4.1 cells.