Andreas M. Beyer

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.

Germ line activation of the Tie2 and SMMHC promoters causes noncell-specific deletion of floxed alleles.

Tissue-specific knockouts generated through Cre-loxP recombination have become an important tool to manipulate the mouse genome. Normally, two successive rounds of breeding are performed to generate mice carrying two floxed target-gene alleles and a transgene expressing Cre-recombinase tissue-specifically. We show herein that two promoters commonly used to generate endothelium-specific (Tie2) and smooth muscle-specific [smooth muscle myosin heavy chain (Smmhc)] knockout mice exhibit activity in the female and male germ lines, respectively. This can result in the inheritance of a null allele in the second generation that is not tissue specific. Careful experimental design is required therefore to ensure that tissue-specific knockouts are indeed tissue specific and that appropriate controls are used to compare strains.

The Human Microcirculation: Regulation of Flow and Beyond

The microcirculation is responsible for orchestrating adjustments in vascular tone to match local tissue perfusion with oxygen demand. Beyond this metabolic dilation, the microvasculature plays a critical role in modulating vascular tone by endothelial release of an unusually diverse family of compounds including nitric oxide, other reactive oxygen species, and arachidonic acid metabolites. Animal models have provided excellent insight into mechanisms of vasoregulation in health and disease. However, there are unique aspects of the human microcirculation that serve as the focus of this review. The concept is put forth that vasculoparenchymal communication is multimodal, with vascular release of nitric oxide eliciting dilation and preserving normal parenchymal function by inhibiting inflammation and proliferation. Likewise, in disease or stress, endothelial release of reactive oxygen species mediates both dilation and parenchymal inflammation leading to cellular dysfunction, thrombosis, and fibrosis. Some pathways responsible for this stress-induced shift in mediator of vasodilation are proposed. This paradigm may help explain why microvascular dysfunction is such a powerful predictor of cardiovascular events and help identify new approaches to treatment and prevention.

Ceramide Changes the Mediator of Flow-Induced Vasodilation from Nitric Oxide to Hydrogen Peroxide in the Human Microcirculation

Rationale: Mitochondrial-derived hydrogen peroxide (H2O2) regulates flow-induced dilation (FID) in microvessels from patients with coronary artery disease. The relationship between ceramide, an independent risk factor for coronary artery disease and a known inducer of mitochondrial reactive oxygen species, and FID is unknown. Objective: We examined the hypothesis that exogenous ceramide induces a switch in the mediator of FID from nitric oxide to H2O2. Methods and Results: Internal diameter changes of resistance arterioles from human adipose and atrial tissue were measured by video microscopy. Mitochondrial H2O2 production was assayed in arterioles using mito peroxy yellow 1. Polyethylene glycol–catalase, rotenone, and Mito-TEMPO impaired FID in healthy adipose arterioles pretreated with ceramide, whereas N&ohgr;-nitro-L-arginine methyl ester had no effect. Mitochondrial H2O2 production was induced in response to flow in healthy adipose vessels pretreated with ceramide, and this was abolished in the presence of polyethylene glycol–catalase. Immunohistochemistry demonstrated ceramide accumulation in arterioles from both healthy patients and patients with coronary artery disease. N&ohgr;-nitro-L-arginine methyl ester reduced vasodilation to flow in adipose as well as atrial vessels from patients with coronary artery disease incubated with GW4869, a neutral sphingomyelinase inhibitor, whereas polyethylene glycol–catalase had no effect. Conclusions: Our data indicate that ceramide has an integral role in the transition of the mediator of FID from nitric oxide to mitochondrial-derived H2O2 and that inhibition of ceramide production can revert the mechanism of dilation back to nitric oxide. Ceramide may be an important target for preventing and treating vascular dysfunction associated with atherosclerosis.

An acute rise in intraluminal pressure shifts the mediator of flow-mediated dilation from nitric oxide to hydrogen peroxide in human arterioles.

Endothelial nitric oxide (NO) is the primary mediator of flow-mediated dilation (FMD) in human adipose microvessels. Impaired NO-mediated vasodilation occurs after acute and chronic hypertension, possibly due to excess generation of reactive oxygen species (ROS). The direct role of pressure elevation in this impairment of human arteriolar dilation is not known. We tested the hypothesis that elevation in pressure is sufficient to impair FMD. Arterioles were isolated from human adipose tissue and cannulated, and vasodilation to graded flow gradients was measured before and after exposure to increased intraluminal pressure (IILP; 150 mmHg, 30 min). The mediator of FMD was determined using pharmacological agents to reduce NO [N(G)-nitro-l-arginine methyl ester (l-NAME), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO)], or H2O2 [polyethylene glycol (PEG)-catalase], and mitochondrial (mt) ROS was quantified using fluorescence microscopy. Exposure to IILP decreased overall FMD (max %dilation: 82.7 ± 4.9 vs. 62 ± 5.6; P < 0.05). This dilation was abolished by treatment with l-NAME prepressure and PEG-catalase after IILP (max %dilation: l-NAME: 23.8 ± 6.1 vs. 74.8 ± 8.6; PEG-catalase: 71.8 ± 5.9 vs. 24.6 ± 10.6). To examine if this change was mediated by mtROS, FMD responses were measured in the presence of the complex I inhibitor rotenone or the mitochondrial antioxidant mitoTempol. Before IILP, FMD was unaffected by either compound; however, both inhibited dilation after IILP. The fluorescence intensity of mitochondria peroxy yellow 1 (MitoPY1), a mitochondria-specific fluorescent probe for H2O2, increased during flow after IILP (%change from static: 12.3 ± 14.5 vs. 127.9 ± 57.7). These results demonstrate a novel compensatory dilator mechanism in humans that is triggered by IILP, inducing a change in the mediator of FMD from NO to mitochondria-derived H2O2.

Critical Role for Telomerase in the Mechanism of Flow Mediated Dilation in the Human Microcirculation

Supplemental Digital Content is available in the text.

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.

In-depth proteomic analysis of human tropomyosin by top-down mass spectrometry

Tropomyosins (Tms) are a family of highly conserved actin-binding proteins that play critical roles in a variety of processes, most notably, in the regulation of muscle contraction and relaxation. It is well known that different Tm isoforms have distinct functions and that altered expression of Tm isoforms could lead to changes in cardiac structure and function. To precisely define Tm isoform expression in the human heart, towards a better understanding of their functional roles, we have employed top-down mass spectrometry for in-depth proteomic characterization of Tm isoforms. Using a minimal amount of human heart tissue from rejected donor organs, we confirmed the presence of multiple Tm isoforms including α-Tm, β-Tm and κ-Tm in the human heart, with α-Tm being the predominant isoform, followed by minor isoforms of β-Tm and κ-Tm. Interestingly, our data revealed regional variations of Tm isoforms and post-translational modifications in the human heart. Specifically, the expression level of κ-Tm was highest in the left atrium but nearly undetectable in the left ventricle. The phosphorylation level of α-Tm (pα-Tm) was significantly higher in the atria than it was in the ventricles. The sequences of all Tm isoforms were characterized and the sites of post-translational modifications were localized. Clearly, top-down mass spectrometry is an attractive method for comprehensive characterization of Tm isoforms and post-translational modifications since it can universally detect and quantify all types of protein modifications without a priori knowledge and without the need for specific antibodies.