Michael P. Massett

ERK5 Activation Inhibits Inflammatory Responses via Peroxisome Proliferator-activated Receptor δ (PPARδ) Stimulation

Peroxisome proliferator-activated receptors (PPAR) decrease the production of cytokine and inducible nitric-oxide synthase (iNOS) expression, which are associated with aging-related inflammation and insulin resistance. Recently, the involvement of the induction of heme oxygenase-1 (HO-1) in regulating inflammation has been suggested, but the exact mechanisms for reducing inflammation by HO-1 remains unclear. We found that overexpression of HO-1 and [Ru(CO)3Cl2]2, a carbon monoxide (CO)-releasing compound, increased not only ERK5 kinase activity, but also its transcriptional activity measured by luciferase assay with the transfection of the Gal4-ERK5 reporter gene. This transcriptional activity is required for coactivation of PPARδ by ERK5 in C2C12 cells. [Ru(CO)3Cl2]2 activated PPARδ transcriptional activity via the MEK5/ERK5 signaling pathway. The inhibition of NF-κB activity by ERK5 activation was reversed by a dominant negative form of PPARδ suggesting that ERK5/PPARδ activation is required for the anti-inflammatory effects of CO and HO-1. Based on these data, we propose a new mechanism by which CO and HO-1 mediate anti-inflammatory effects via activating ERK5/PPARδ, and ERK5 mediates CO and HO-1-induced PPARδ activation via its interaction with PPARδ.

Effect of heating on the hemodynamic responses to vasoactive agents

During hyperthermia, vasoconstrictor tone in the viscera is lost despite high levels of sympathetic neural outflow and plasma catecholamines, suggesting that vascular responsiveness to adrenergic receptor stimulation is reduced. The purpose of this study was to determine whether adrenoceptor-mediated control of vascular resistance is altered at high body core temperatures. The hemodynamic responses to adrenoceptor agonists were examined in chloralose-anesthetized rats heated to colonic temperatures (Tco) of 37, 39, and 41.5 degrees C. Elevating Tco to 39 degrees C did not alter the hemodynamic responses to any of these agents. Further heating to 41.5 degrees C markedly attenuated the hemodynamic responses to alpha- and beta-adrenoceptor agonists. Similarly, the regional and systemic hemodynamic responses to ANG II and endothelin were also reduced at 41.5 degrees C. In contrast, the hemodynamic responses to endothelium-dependent and -independent vasodilator agents were unchanged or slightly reduced at 41.5 degrees C. The blunted hemodynamic responses observed at 41.5 degrees C indicate that vascular reactivity to vasoconstrictor agents is reduced with hyperthermia and suggest that this nonspecific change in vascular responsiveness may contribute the circulatory collapse associated with high body temperatures.During hyperthermia, vasoconstrictor tone in the viscera is lost despite high levels of sympathetic neural outflow and plasma catecholamines, suggesting that vascular responsiveness to adrenergic receptor stimulation is reduced. The purpose of this study was to determine whether adrenoceptor-mediated control of vascular resistance is altered at high body core temperatures. The hemodynamic responses to adrenoceptor agonists were examined in chloralose-anesthetized rats heated to colonic temperatures (Tco) of 37, 39, and 41.5°C. Elevating Tco to 39°C did not alter the hemodynamic responses to any of these agents. Further heating to 41.5°C markedly attenuated the hemodynamic responses to α- and β-adrenoceptor agonists. Similarly, the regional and systemic hemodynamic responses to ANG II and endothelin were also reduced at 41.5°C. In contrast, the hemodynamic responses to endothelium-dependent and -independent vasodilator agents were unchanged or slightly reduced at 41.5°C. The blunted hemodynamic responses observed at 41.5°C indicate that vascular reactivity to vasoconstrictor agents is reduced with hyperthermia and suggest that this nonspecific change in vascular responsiveness may contribute the circulatory collapse associated with high body temperatures.

Angiotensin II Type 2 Receptor Expression After Vascular Injury: Differing Effects of Angiotensin-Converting Enzyme Inhibition and Angiotensin Receptor Blockade

It has been suggested that the effects of angiotensin II type 1 receptor (AT1R) blockers are in part because of angiotensin II type 2 receptor (AT2R) signaling. Interactions between the AT2R and kinins modulate cardiovascular function. Because AT2R expression increases after vascular injury, we hypothesized that the effects on vascular remodeling of the AT1R blocker valsartan and the ACE inhibitor benazepril require AT2R signaling through the bradykinin 1 and 2 receptors (B1R and B2R). To test this hypothesis, Brown Norway rats were assigned to 8 treatments (n=16): valsartan, valsartan+PD123319 (AT2R inhibitor), valsartan+des-arg9-[Leu8]-bradykinin (B1R inhibitor), valsartan+HOE140 (B2R inhibitor), benazepril, benazepril+HOE140, amlodipine, and vehicle. After 1 week of treatment, carotid balloon injury was performed. Two weeks later, carotids were harvested for morphometry and analysis of receptor expression by immunohistochemistry and Western blotting. Valsartan and benazepril significantly reduced the intima:media ratio compared with vehicle. Blockade of AT2R, B1R, or B2R in the presence of valsartan prevented the reduction seen with valsartan alone. B2R blockade inhibited the effect of benazepril. Injury increased AT1R, AT2R, B1R, and B2R expression. Treatment with valsartan but not benazepril significantly increased intima AT2R expression 2-fold compared with vehicle, which was not reversed by inhibition of AT2R, B1R, and B2R. Functionally, valsartan increased intimal cGMP levels compared with vehicle, and this increase was inhibited by blocking the AT2R, B1R, and B2R. Results suggest that AT2R expression and increased cGMP represent a molecular mechanism that differentiates AT1R blockers, such as valsartan, from angiotensin-converting enzyme inhibitors like benazepril.

Axl Mediates Vascular Remodeling Induced by Deoxycorticosterone Acetate–Salt Hypertension

Axl, a receptor tyrosine kinase, was recently identified as a novel candidate gene in a genetic model of salt-sensitive hypertension (Sabra rat). Our group first reported that Axl plays a significant role in vascular remodeling in response to injury. Here we investigated the role of Axl in the pathogenesis of hypertension in a deoxycorticosterone acetate (DOCA)–salt model. Hypertension was induced in Axl wild-type (Axl+/+) mice and Axl-deficient (Axl−/−) mice by uninephrectomy and DOCA-salt for 6 weeks. Controls were uninephrectomized and received tap water and regular chow ad libitum. DOCA-salt treatment increased systolic blood pressure by 25 mm Hg in both genotypes after 1 week. Systolic blood pressure remained significantly elevated in Axl+/+ DOCA, whereas systolic blood pressure levels in Axl−/− DOCA mice were the same as controls at 6 weeks. DOCA-salt increased relative kidney weight and glomerular hypertrophy by 40% compared with controls in both genotypes. Consistent with levels of systolic blood pressure, endothelium-dependent vasorelaxation was impaired in Axl+/+ DOCA mice compared with Axl+/+ controls, whereas in Axl−/− DOCA mice relaxation responses were similar to Axl−/− controls. In addition, endothelium-independent vasorelaxation was improved in Axl−/− DOCA mice compared with Axl+/+ DOCA mice. Nitrotyrosine and phospho-Akt immunoreactivity was significantly reduced in arteries from Axl−/− DOCA mice compared with Axl+/+ DOCA mice. The remodeling index of the mesenteric artery (media:lumen ratio) was significantly increased in Axl+/+ DOCA mice compared with Axl−/− DOCA mice. Finally, increased vascular apoptosis in the Axl−/− DOCA mice suggests a likely mechanism for Axl-dependent effects on hypertension. These data strengthen the pathogenic role for Axl in salt-sensitive hypertension.

G-Protein–Coupled Receptor Kinase Interacting Protein-1 Is Required for Pulmonary Vascular Development

Background— The G-protein–coupled receptor kinase interacting protein-1 (GIT1) is a multidomain scaffold protein that participates in many cellular functions including receptor internalization, focal adhesion remodeling, and signaling by both G-protein–coupled receptors and tyrosine kinase receptors. However, there have been no in vivo studies of GIT1 function to date. Methods and Results— To determine essential functions of GIT1 in vivo, we generated a traditional GIT1 knockout mouse. GIT1 knockout mice exhibited ≈60% perinatal mortality. Pathological examination showed that the major abnormality in GIT1 knockout mice was impaired lung development characterized by markedly reduced numbers of pulmonary blood vessels and increased alveolar spaces. Given that vascular endothelial growth factor (VEGF) is essential for pulmonary vascular development, we investigated the role of GIT1 in VEGF signaling in the lung and cultured endothelial cells. Because activation of phospholipase-Cγ (PLCγ) and extracellular signal-regulated kinases 1/2 (ERK1/2) by angiotensin II requires GIT1, we hypothesized that GIT1 mediates VEGF-dependent pulmonary angiogenesis by modulating PLCγ and ERK1/2 activity in endothelial cells. In cultured endothelial cells, knockdown of GIT1 decreased VEGF-mediated phosphorylation of PLCγ and ERK1/2. PLCγ and ERK1/2 activity in lungs from GIT1 knockout mice was reduced postnatally. Conclusions— Our data support a critical role for GIT1 in pulmonary vascular development by regulating VEGF-induced PLCγ and ERK1/2 activation.

GIT1 Mediates HDAC5 Activation by Angiotensin II in Vascular Smooth Muscle Cells

Objective—The G protein–coupled receptor (GPCR)-kinase2 interacting protein1 (GIT1) is a scaffold protein involved in angiotensin II (Ang II) signaling. Histone deacetylase-5 (HDAC5) has emerged as an important substrate of calcium/calmodulin-dependent protein kinase II (CamK II) in GPCR signaling. Here we investigated the hypothesis that Ang II–mediated vascular smooth muscle cell (VSMC) gene transcription involves GIT1-CamK II–dependent phosphorylation of HDAC5. Methods and Results—Ang II rapidly stimulated phosphorylation of HDAC5 at Ser498 in VSMCs. Knockdown of GIT1 significantly decreased HDAC5 phosphorylation induced by Ang II. The involvement of Src, phospholipase &ggr; (PLC&ggr;), and CamK II in GIT1-mediated HDAC5 phosphorylation was demonstrated. The association of GIT1 and CamK II was constitutive but increased after stimulation with Ang II. Moreover, the interaction of GIT1 and CamK II through the ARF GTPase-activating protein (ARF-GAP) and coiled-coil domains of GIT1 was essential for the phosphorylation of HDAC5. Finally, knockdown of GIT1 decreased myocyte enhancer factor 2 transcriptional activity induced by Ang II. Conclusions—This study identifies a novel function for GIT1 as a mediator of Ang II–induced VSMC gene transcription via a Src-PLC&ggr;-CamK II-HDAC5 signaling pathway.

Mice Lacking TR4 Nuclear Receptor Develop Mitochondrial Myopathy with Deficiency in Complex I

The estimated incidence of mitochondrial diseases in humans is approximately 1:5000 to 1:10,000, whereas the molecular mechanisms for more than 50% of human mitochondrial disease cases still remain unclear. Here we report that mice lacking testicular nuclear receptor 4 (TR4(-/-)) suffered mitochondrial myopathy, and histological examination of TR4(-/-) soleus muscle revealed abnormal mitochondrial accumulation. In addition, increased serum lactate levels, decreased mitochondrial ATP production, and decreased electron transport chain complex I activity were found in TR4(-/-) mice. Restoration of TR4 into TR4(-/-) myoblasts rescued mitochondrial ATP generation capacity and complex I activity. Further real-time PCR quantification and promoter studies found TR4 could modulate complex I activity via transcriptionally regulating the complex I assembly factor NDUFAF1, and restoration of NDUFAF1 level in TR4(-/-) myoblasts increased mitochondrial ATP generation capacity and complex I activity. Together, these results suggest that TR4 plays vital roles in mitochondrial function, which may help us to better understand the pathogenesis of mitochondrial myopathy, and targeting TR4 via its ligands/activators may allow us to develop better therapeutic approaches.

Impaired Vasorelaxation in Inbred Mice Is Associated with Alterations in Both Nitric Oxide and Super Oxide Pathways

Recently, we showed that genetic factors determine flow-dependent vascular remodeling. Among five inbred mouse strains, the SJL strain developed the largest intima in response to low flow. Because SJL mice have a spontaneous mutation in superoxide dismutase 2 (SOD-2) we tested the hypothesis that strain-specific variations in vascular function are due to alterations in redox and nitric oxide (NO) pathways. Vasorelaxation to acetylcholine was significantly impaired in aortic rings from SJL compared to C3H or FVB mice (up to 40%). Relaxation to the endothelium-independent vasodilator sodium nitroprusside (SNP) in SJL mice was also significantly impaired at low concentrations, with decreases in sensitivity and maximal relaxation to SNP compared to C3H and FVB mice. Western blot analyses showed significantly decreased expression (∼40%) of eNOS, PKG and SOD-2 proteins in SJL vasculature compared to C3H. Intact aortas from SJL showed significantly increased nitrotyrosine and decreased SOD-2 expression compared to C3H by immunohistochemistry. Basal levels of superoxide in aortas from SJL were not significantly different than C3H as measured by dihydroethidine. In summary, relatively small alterations in redox (SOD-2) and NO pathways (eNOS and PKG) may contribute to significantly impaired vasorelaxation in SJL mice.

Identification of exercise capacity QTL using association mapping in inbred mice

There are large interindividual differences in exercise capacity. It is well established that there is a genetic basis for these differences. However, the genetic factors underlying this variation are undefined. Therefore, the purpose of this study was to identify novel putative quantitative trait loci (QTL) for exercise capacity by measuring exercise capacity in inbred mice and performing genome-wide association mapping. Exercise capacity, defined as run time and work, was assessed in male mice (n = 6) from 34 strains of classical and wild-derived inbred mice performing a graded treadmill test. Genome-wide association mapping was performed with an efficient mixed-model association (EMMA) algorithm to identify QTL. Exercise capacity was significantly different across strains. Run time varied by 2.7-fold between the highest running strain (C58/J) and the lowest running strain (A/J). These same strains showed a 16.5-fold difference in work. Significant associations were identified for exercise time on chromosomes 1, 2, 7, 11, and 13. The QTL interval on chromosome 2 (~168 Mb) contains one gene, Nfatc2, and overlaps with a suggestive QTL for training responsiveness in humans. These results provide phenotype data on the widest range of inbred strains tested thus far and indicate that genetic background significantly influences exercise capacity. Furthermore, the novel QTLs identified in the current study provide new targets for investigating the underlying mechanisms for variation in exercise capacity.

Quantitative trait loci for exercise training responses in FVB/NJ and C57BL/6J mice

The genetic factors determining the magnitude of the response to exercise training are poorly understood. The aim of this study was to identify quantitative trait loci (QTL) associated with adaptation to exercise training in a cross between FVB/NJ (FVB) and C57BL/6J (B6) mice. Mice completed an exercise performance test before and after a 4-wk treadmill running program, and changes in exercise capacity, expressed as work (kg.m), were calculated. Changes in work in F(2) mice averaged 1.51 +/- 0.08 kg.m (94.3 +/- 7.3%), with a range of -1.67 to +4.55 kg.m. All F(2) mice (n = 188) were genotyped at 20-cM intervals with 103 single nucleotide polymorphisms (SNPs), and genomewide linkage scans were performed for pretraining, posttraining, and change in work. Significant QTL for pretraining work were located on chromosomes 14 at 4.0 cM [3.72 logarithm of odds (LOD)] and 19 at 34.4 cM (3.63 LOD). For posttraining work significant QTL were located on chromosomes 3 at 60 cM (4.66 LOD) and 14 at 26 cM (4.99 LOD). Suggestive QTL for changes in work were found on chromosomes 11 at 44.6 cM (2.30 LOD) and 14 at 36 cM (2.25 LOD). When pretraining work was used as a covariate, a potential QTL for change in work was identified on chromosome 6 at 68 cM (3.56 LOD). These data indicate that one or more QTL determine exercise capacity and training responses in mice. Furthermore, these data suggest that the genes that determine pretraining work and training responses may differ.