Henry L. Keen

Interference with PPARγ Function in Smooth Muscle Causes Vascular Dysfunction and Hypertension

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that plays a critical role in metabolism. Thiazolidinediones, high-affinity PPARgamma ligands used clinically to treat type II diabetes, have been reported to lower blood pressure and provide other cardiovascular benefits. Some mutations in PPARgamma (PPARG) cause type II diabetes and severe hypertension. Here we tested the hypothesis that PPARgamma in vascular muscle plays a role in the regulation of vascular tone and blood pressure. Transgenic mice expressing dominant-negative mutations in PPARgamma under the control of a smooth-muscle-specific promoter exhibit a loss of responsiveness to nitric oxide and striking alterations in contractility in the aorta, hypertrophy and inward remodeling in the cerebral microcirculation, and systolic hypertension. These results identify PPARgamma as pivotal in vascular muscle as a regulator of vascular structure, vascular function, and blood pressure, potentially explaining some of the cardioprotective effects of thiazolidinediones.

Interference With PPARγ Signaling Causes Cerebral Vascular Dysfunction, Hypertrophy, and Remodeling

The transcription factor PPARγ is expressed in endothelium and vascular muscle where it may exert antiinflammatory and antioxidant effects. We tested the hypothesis that PPARγ plays a protective role in the vasculature by examining vascular structure and function in heterozygous knockin mice expressing the P465L dominant negative mutation in PPARγ (L/+). In L/+ aorta, responses to the endothelium-dependent agonist acetylcholine (ACh) were not affected, but there was an increase in contraction to serotonin, PGF2α, and endothelin-1. In cerebral blood vessels both in vitro and in vivo, ACh produced dilation that was markedly impaired in L/+ mice. Superoxide levels were elevated in cerebral arterioles from L/+ mice and responses to ACh were restored to normal with a scavenger of superoxide. Diameter of maximally dilated cerebral arterioles was less, whereas wall thickness and cross-sectional area was greater in L/+ mice, indicating cerebral arterioles underwent hypertrophy and remodeling. Thus, interference with PPARγ signaling produces endothelial dysfunction via a mechanism involving oxidative stress and causes vascular hypertrophy and inward remodeling. These findings indicate that PPARγ has vascular effects which are particularly profound in the cerebral circulation and provide genetic evidence that PPARγ plays a critical role in protecting blood vessels.

Endothelium-Specific Interference With Peroxisome Proliferator Activated Receptor Gamma Causes Cerebral Vascular Dysfunction in Response to a High-Fat Diet

The ligand-activated transcription factor peroxisome proliferator activated receptor gamma (PPAR&ggr;) 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 PPAR&ggr; under the control of an endothelial-specific promoter to test the hypothesis that endothelial PPAR&ggr; 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 PPAR&ggr; 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 PPAR&ggr;, 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 PPAR&ggr; 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 PPAR&ggr; plays a critical role in protecting blood vessels in response to a high-fat diet.

Insulin-Induced Hypertension in Rats Depends on an Intact Renin-Angiotensin System

This study tested the dependence of insulin-induced hypertension in rats on a functional renin-angiotensin system. Rats were instrumented with chronic artery and vein catheters and housed in metabolic cages. After acclimation, 10 rats began receiving the angiotensin-converting enzyme inhibitor (ACEI) benazepril at 1.8 mg.kg-1.d-1 via a continuous intravenous infusion that was maintained throughout the study; 8 control rats received vehicle. Four days after starting ACEI or vehicle, all rats entered a 5-day control period that was followed by a 7-day insulin infusion at 1.5 mU.kg-1.min-1. Glucose was coinfused at 22 mg.kg-1.min-1 to prevent hypoglycemia. Insulin infusion in control rats increased mean arterial pressure (MAP; measured 24 h/d) from an average of 101 +/- 1 to 113 +/- 2 mm Hg on day 1; MAP averaged 110 +/- 1 mm Hg for the 7-day infusion period. Glomerular filtration rate decreased, although not significantly, from 2.7 +/- 0.1 to 2.1 +/- 0.2 mL/min on day 3. Chronic ACEI decreased baseline MAP from an average of 97 +/- 1 to 79 +/- 1 mm Hg and markedly attenuated the increase in MAP during insulin. MAP averaged 81 +/- 1 mm Hg for the 7-day period and increased significantly, to 85 +/- 2 mm Hg, only on day 3. Likewise, the tendency for glomerular filtration rate to decrease was blunted. These results indicate that insulin-induced hypertension in rats depends on angiotensin II and suggest that a reduction in glomerular filtration rate contributes to the shift in pressure natriuresis.

Renal proximal tubule angiotensin AT1A receptors regulate blood pressure

All components of the renin angiotensin system necessary for ANG II generation and action have been reported to be present in renal proximal convoluted tubules. Given the close relationship between renal sodium handling and blood pressure regulation, we hypothesized that modulating the action of ANG II specifically in the renal proximal tubules would alter the chronic level of blood pressure. To test this, we used a proximal tubule-specific, androgen-dependent, promoter construct (KAP2) to generate mice with either overexpression of a constitutively active angiotensin type 1A receptor transgene or depletion of endogenous angiotensin type 1A receptors. Androgen administration to female transgenic mice caused a robust induction of the transgene in the kidney and increased baseline blood pressure. In the receptor-depleted mice, androgen administration to females resulted in a Cre recombinase-mediated deletion of angiotensin type 1A receptors in the proximal tubule and reduced blood pressure. In contrast to the changes observed at baseline, there was no difference in the blood pressure response to a pressor dose of ANG II in either experimental model. These data, from two separate mouse models, provide evidence that ANG II signaling via the type 1A receptor in the renal proximal tubule is a regulator of systemic blood pressure under baseline conditions.

Does Peroxisome Proliferator-activated Receptor-γ (PPARγ) Protect from Hypertension Directly through Effects in the Vasculature?

Peroxisome proliferator-activated receptor-γ (PPARγ) is a ligand-activated transcription factor of the nuclear hormone receptor superfamily. Increasing evidence suggests that PPARγ is involved in the regulation of vascular function and blood pressure in addition to its well recognized role in metabolism. Thiazolidinediones, PPARγ agonists, lower blood pressure and have protective vascular effects through largely unknown mechanisms. In contrast, loss-of-function dominant-negative mutations in human PPARγ cause insulin resistance and severe early onset hypertension. Recent studies using genetically manipulated mouse models have begun to specifically address the importance of PPARγ in the vasculature. In this minireview, evidence for a protective role of PPARγ in the endothelium and vascular smooth muscle, derived largely from studies of genetically manipulated mice, will be discussed.

PPARγ Regulates Resistance Vessel Tone Through a Mechanism Involving RGS5-Mediated Control of Protein Kinase C and BKCa Channel Activity

Rationale: Activation of peroxisome proliferator−activated receptor-&ggr; (PPAR&ggr;) by thiazolidinediones lowers blood pressure, whereas PPAR&ggr; mutations cause hypertension. Previous studies suggest these effects may be mediated through the vasculature, but the underlying mechanisms remain unclear. Objective: To identify PPAR&ggr; mechanisms and transcriptional targets in vascular smooth muscle and their role in regulating resistance artery tone. Methods and Results: We studied mesenteric artery (MA) from transgenic mice expressing dominant-negative (DN) mutant PPAR&ggr; driven by a smooth muscle cell−specific promoter. MA from transgenic mice exhibited a robust increase in myogenic tone. Patch clamp analysis revealed a reduced large conductance Ca2+-activated K+ (BKCa) current in freshly dissociated smooth muscle cell from transgenic MA. Inhibition of protein kinase C corrected both enhanced myogenic constriction and impaired the large conductance Ca2+-activated K+ channel function. Gene expression profiling revealed a marked loss of the regulator of G protein signaling 5 (RGS5) mRNA in transgenic MA, which was accompanied by a substantial increase in angiotensin II–induced constriction in MA. Small interfering RNA targeting RGS5 caused augmented myogenic tone in intact mesenteric arteries and increased activation of protein kinase C in smooth muscle cell cultures. PPAR&ggr; and PPAR&dgr; each bind to a PPAR response element close to the RGS5 promoter. RGS5 expression in nontransgenic MA was induced after activation of either PPAR&ggr; or PPAR&dgr;, an effect that was markedly blunted by DN PPAR&ggr;. Conclusions: We conclude that RGS5 in smooth muscle is a PPAR&ggr; and PPAR&dgr; target, which when activated blunts angiotensin II–mediated activation of protein kinase C, and preserves the large conductance Ca2+-activated K+ channel activity, thus providing tight control of myogenic tone in the microcirculation.

Dominant negative PPARγ promotes atherosclerosis, vascular dysfunction, and hypertension through distinct effects in endothelium and vascular muscle

Agonists of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) have potent insulin-sensitizing effects and inhibit atherosclerosis progression in patients with Type II diabetes. Conversely, missense mutations in the ligand-binding domain of PPARγ that render the transcription factor dominant negative (DN) cause early-onset hypertension and Type II diabetes. We tested the hypothesis that DN PPARγ-mediated interference of endogenous wild-type PPARγ in the endothelium and vascular smooth muscle exacerbates atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-specific expression of DN PPARγ on the ApoE(-/-) background unmasked significant impairment of endothelium-dependent relaxation in aortic rings, increased systolic blood pressure, altered expression of atherogenic markers (e.g., Cd36, Mcp1, Catalase), and enhanced diet-induced atherosclerotic lesion formation in aorta. Smooth muscle-specific expression of DN PPARγ, which induces aortic dysfunction and increased systolic blood pressure at baseline, also resulted in enhanced diet-induced atherosclerotic lesion formation in aorta on the ApoE(-/-) background that was associated with altered expression of a shared, yet distinct, set of atherogenic markers (e.g., Cd36, Mcp1, Osteopontin, Vcam1). In particular, induction of Osteopontin expression by smooth muscle-specific DN PPARγ correlated with increased plaque calcification. These data demonstrate that inhibition of PPARγ function specifically in the vascular endothelium or smooth muscle may contribute to cardiovascular disease.

Bioinformatic Analysis of Gene Sets Regulated by Ligand-Activated and Dominant-Negative Peroxisome Proliferator–Activated Receptor γ in Mouse Aorta

Objective—Drugs that activate peroxisome proliferator–activated receptor (PPAR) &ggr; improve glucose sensitivity and lower blood pressure, whereas dominant-negative mutations in PPAR&ggr; cause severe insulin resistance and hypertension. We hypothesize that these PPAR&ggr; mutants regulate target genes opposite to those of ligand-mediated activation, and we tested this hypothesis on a genomewide scale. Methods and Results—We integrated gene expression data in aorta specimens from mice treated with the PPAR&ggr; ligand rosiglitazone with data from mice containing a globally expressed knockin of the PPAR&ggr; P465L dominant-negative mutation. We also integrated our data with publicly available data sets containing the following: (1) gene expression profiles in many human tissues, (2) PPAR&ggr; target genes in 3T3-L1 adipocytes, and (3) experimentally validated PPAR&ggr; binding sites throughout the genome. Many classic PPAR&ggr; target genes were induced by rosiglitazone and repressed by dominant-negative PPAR&ggr;. A similar pattern was observed for about 90% of the gene sets regulated by both rosiglitazone and dominant-negative PPAR&ggr;. Genes exhibiting this pattern of contrasting regulation were significantly enriched for nearby PPAR&ggr; binding sites. Conclusion—These results provide convincing evidence that the PPAR&ggr; P465L mutation causes transcriptional effects that are opposite to those mediated by PPAR&ggr; ligand, thus validating mice carrying the mutation as a model of PPAR&ggr; interference.

Regulation of Renin Gene Expression by Oxidative Stress

Increased arterial pressure, angiotensin II, and cytokines each result in feedback inhibition of renin gene expression. Because angiotensin II and cytokines can stimulate reactive oxygen species production, we tested the hypothesis that oxidative stress may be a mediator of this inhibition. Treatment of renin-expressing As4.1 cells with the potent cytokine tumor necrosis factor-&agr; caused an increase in the steady-state levels of cellular reactive oxygen species, which was reversed by the antioxidant N-acetylcysteine. Exogenous H2O2 caused a dose- and time-dependent decrease in the level of endogenous renin mRNA and decreased the transcriptional activity of a 4.1-kb renin promoter fused to luciferase, which was maximal when the renin enhancer was present. The effect of H2O2 appeared to be specific to renin, because there was no change in the expression of &bgr;-actin or cyclophilin mRNA or transcriptional activity of the SV40 promoter. The tumor necrosis factor-&agr;–induced decrease in renin mRNA was partially reversed by either N-acetylcysteine or panepoxydone, a nuclear factor &kgr;B (NF&kgr;B) inhibitor. Interestingly, H2O2 did not induce NF&kgr;B in As4.1 cells, and panepoxydone had no effect on the downregulation of renin mRNA by H2O2. The transcriptional activity of a cAMP response element-luciferase construct was decreased by both tumor necrosis factor-&agr; and H2O2. These data suggest that cellular reactive oxygen species can negatively regulate renin gene expression via an NF&kgr;B-independent mechanism involving the renin enhancer and inhibiting cAMP response element–mediated transcription. Our data further suggest that tumor necrosis factor-&agr; decreases renin expression through both NF&kgr;B-dependent and NF&kgr;B-independent mechanisms, the latter involving the production of reactive oxygen species.