Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications.

There is strong evidence that overproduction of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta is a major cause of vascular dysfunction in preeclampsia through sFLT1-dependent antagonism of VEGF. However, the cause of placental sFLT1 upregulation is not known. Here we demonstrated that in women with preeclampsia, sFLT1 is upregulated in placental trophoblasts, while VEGF is upregulated in adjacent maternal decidual cells. In response to VEGF, expression of sFlt1 mRNA, but not full-length Flt1 mRNA, increased in cultured murine trophoblast stem cells. We developed a method for transgene expression specifically in mouse endometrium and found that endometrial-specific VEGF overexpression induced placental sFLT1 production and elevated sFLT1 levels in maternal serum. This led to pregnancy losses, placental vascular defects, and preeclampsia-like symptoms, including hypertension, proteinuria, and glomerular endotheliosis in the mother. Knockdown of placental sFlt1 with a trophoblast-specific transgene caused placental vascular changes that were consistent with excess VEGF activity. Moreover, sFlt1 knockdown in VEGF-overexpressing animals enhanced symptoms produced by VEGF overexpression alone. These findings indicate that sFLT1 plays an essential role in maintaining vascular integrity in the placenta by sequestering excess maternal VEGF and suggest that a local increase in VEGF can trigger placental overexpression of sFLT1, potentially contributing to the development of preeclampsia and other pregnancy complications.

FXR agonist INT-747 upregulates DDAH expression and enhances insulin sensitivity in high-salt fed Dahl rats.

AIMS: Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity. METHODS AND RESULTS: In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls. CONCLUSION: Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.

Regulatory T cells limit vascular endothelial injury and prevent pulmonary hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is an incurable disease associated with viral infections and connective tissue diseases. The relationship between inflammation and disease pathogenesis in these disorders remains poorly understood. OBJECTIVE: To determine whether immune dysregulation due to absent T-cell populations directly contributes to the development of PAH. METHODS AND RESULTS: Vascular endothelial growth factor receptor 2 (VEGFR2) blockade induced significant pulmonary endothelial apoptosis in T-cell-deficient rats but not in immune-reconstituted (IR) rats. T cell-lymphopenia in association with VEGFR2 blockade resulted in periarteriolar inflammation with macrophages, and B cells even prior to vascular remodeling and elevated pulmonary pressures. IR prevented early inflammation and attenuated PAH development. IR with either CD8 T cells alone or with CD4-depleted spleen cells was ineffective in preventing PAH, whereas CD4-depleting immunocompetent euthymic animals increased PAH susceptibility. IR with either CD4(+)CD25(hi) or CD4(+)CD25(-) T cell subsets prior to vascular injury attenuated the development of PAH. IR limited perivascular inflammation and endothelial apoptosis in rat lungs in association with increased FoxP3(+), IL-10- and TGF-ß-expressing CD4 cells, and upregulation of pulmonary bone morphogenetic protein receptor type 2 (BMPR2)-expressing cells, a receptor that activates endothelial cell survival pathways. CONCLUSIONS: PAH may arise when regulatory T-cell (Treg) activity fails to control endothelial injury. These studies suggest that regulatory T cells normally function to limit vascular injury and may protect against the development of PAH.

Disruption of the apelin-APJ system worsens hypoxia-induced pulmonary hypertension.

OBJECTIVE: The G-protein-coupled receptor APJ and its ligand apelin are highly expressed in the pulmonary vasculature, but their function in this vascular bed is unclear. We hypothesized that disruption of apelin signaling would lead to worsening of the vascular remodeling associated with pulmonary hypertension (PH). METHODS AND RESULTS: We found that apelin-null mice developed more severe PH compared with wild-type mice when exposed to chronic hypoxia. Micro-computed tomography of the pulmonary arteries demonstrated significant pruning of the microvasculature in the apelin-null mice. Apelin-null mice had a significant reduction of serum nitrate levels. This was secondary to downregulation of endothelial nitric oxide synthase (eNOS), which was associated with reduced expression of Kruppel-like factor 2 (KLF2), a known regulator of eNOS expression. In vitro knockdown studies targeting apelin in human pulmonary artery endothelial cells demonstrated decreased eNOS and KLF2 expression, as well as impaired phosphorylation of AMP-activated kinase and eNOS. Moreover, serum apelin levels of patients with PH were significantly lower than those of controls. CONCLUSIONS: These data demonstrate that disruption of apelin signaling can exacerbate PH mediated by decreased activation of AMP-activated kinase and eNOS, and they identify this pathway as a potentially important therapeutic target for treatment of this refractory human disease.

Gene expression profiling: classification of mice with left ventricle systolic dysfunction using microarray analysis.

OBJECTIVE: We tested the hypothesis that a set of differentially expressed genes could be used to classify mice according to cardiovascular phenotype after prolonged catecholamine stress. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: One hundred seventy-three male mice were studied: wild-type (WT) C57, WT FVB, WT B6129SF2/J, and beta2 adrenergic receptor knockout. INTERVENTIONS: Mice of each genotype were randomly assigned to 14-day infusions of isoproterenol (120 microg/g/day) or no treatment. Approximately half of the animals underwent left ventricle pressure volume loop analysis. The remaining animals were killed for extraction of messenger RNA from whole heart preparations for microarray analysis. MEASUREMENTS AND MAIN RESULTS: We observed that WT FVB and beta2 adrenergic receptor knockout mice developed systolic dysfunction in response to continuous catecholamine infusion, whereas WT C57 mice developed diastolic dysfunction. Using these mice as the derivation cohort, we identified a set of 83 genes whose differential expression correlated with left ventricle systolic dysfunction. The gene set was then used to accurately classify mice from a separate group (WT B6129SF2/J) into the cohort that developed left ventricle systolic dysfunction after catecholamine stress. CONCLUSIONS: The differential expression pattern of 83 genes can be used to accurately classify mice according to physiological phenotype after catecholamine stress.

Postischemic brain injury is attenuated in mice lacking the beta2-adrenergic receptor.

BACKGROUND: Several beta-adrenergic receptor (betaAR) antagonists have been shown to have neuroprotective effects against cerebral ischemia. However, clenbuterol, a beta(2)AR agonist, was shown to have neuroprotective activity by increasing nerve growth factor expression. We used beta(2)AR knockout mice and a beta(2) selective antagonist to test the effect of loss of beta(2)ARs on outcome from transient focal cerebral ischemia. METHODS: Ischemia was induced by the intraluminal suture method, for 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. Neurological score was determined at 24 h reperfusion and infarct size was determined by cresyl violet or 2,3,5-triphenyltetrazolium chloride staining. beta(2)AR knockout mice and wild-type congenic FVB/N controls were studied, as well as 2 groups of wild type mice given either ICI 118,551 (0.2 mg/kg) or 0.9% saline intraperitoneally 30 min before MCAO (n = 10 per group). Changes in expression of heat shock protein (Hsp)72 after ischemia were examined by immunohistochemistry and western blots. RESULTS: Compared with wild type littermates, infarct volume was decreased by 22.3% in beta(2)AR knockout mice (39.7 +/- 10.7 mm(3) vs 51.0 +/- 11.4 mm(3), n = 10/group, P = 0.034) after 60 min of MCAO followed by 24 h reperfusion. Pretreatment with a beta(2)AR selective antagonist, ICI 118,551, also decreased infarct size significantly, by 25.1%, compared with the saline control (32.8 +/- 11.9 mm(3) vs 43.8 +/- 10.3 mm(3), n = 10/group, P = 0.041). Neurological scores were also significantly improved in mice lacking the beta(2)AR or pretreated with ICI 118,551. After cerebral ischemia, total levels of Hsp72 and the number of Hsp72 immunopositive cells were greater in mice lacking beta(2) AR. CONCLUSION: Brain injury is reduced and neurological outcome improved after MCAO in mice lacking the beta(2)AR, or in wild type mice pretreated with a selective beta(2)AR antagonist. This is consistent with a shift away from prosurvival signaling to prodeath signaling in the presence of beta(2)AR activation in cerebral ischemia. Protection is associated with higher levels of Hsp72, a known antideath protein. The effect of beta(2)AR signaling in the setting of cerebral ischemia is complex and warrants further study.

Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis.

Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function and a vasodepressor function in the systemic circulation. The apelin-APJ pathway appears to have opposing physiological roles to the renin-angiotensin system. Here we investigated whether the apelin-APJ pathway can directly antagonize vascular disease-related Ang II actions. In ApoE-KO mice, exogenous Ang II induced atherosclerosis and abdominal aortic aneurysm formation; we found that coinfusion of apelin abrogated these effects. Similarly, apelin treatment rescued Ang II-mediated increases in neointimal formation and vascular remodeling in a vein graft model. NO has previously been implicated in the vasodepressor function of apelin; we found that apelin treatment increased NO bioavailability in ApoE-KO mice. Furthermore, infusion of an NO synthase inhibitor blocked the apelin-mediated decrease in atherosclerosis and aneurysm formation. In rat primary aortic smooth muscle cells, apelin inhibited Ang II-mediated transcriptional regulation of multiple targets as measured by reporter assays. In addition, we demonstrated by coimmunoprecipitation and fluorescence resonance energy transfer analysis that the Ang II and apelin receptors interacted physically. Taken together, these findings indicate that apelin signaling can block Ang II actions in vascular disease by increasing NO production and inhibiting Ang II cellular signaling.

Signaling from beta1- and beta2-adrenergic receptors is defined by differential interactions with PDE4.

Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that beta1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a beta2AR/beta-arrestin/PDE complex reported previously. The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.

The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo.

OBJECTIVE: The endogenous peptide apelin is differentially regulated in cardiovascular disease but the nature of its role in cardiac function remains unclear. METHODS: We investigated the functional relevance of this peptide using ECG and respiration gated magnetic resonance imaging, conductance catheter pressure-volume hemodynamic measurements, and echocardiography in vivo. In addition, we carried out histology and immunohistochemistry to assess cardiac hypertrophy and to localize apelin and APJ in the adult and embryonic mouse heart. RESULTS: Intraperitoneal injection of apelin (300 microg/kg) resulted in a decrease in left ventricular end diastolic area (pre: 0.122+/-0.007; post: 0.104+/-0.005 cm(2), p=0.006) and an increase in heart rate (pre: 537+/-20; post: 559+/-19 beats per minute, p=0.03). Hemodynamic measurements revealed a marked increase in ventricular elastance (pre: 3.7+/-0.9; post: 6.5+/-1.4 mm Hg/RVU, p=0.018) and preload recruitable stroke work (pre: 27.4+/-8.0; post: 51.8+/-3.1, p=0.059) with little change in diastolic parameters following acute infusion of apelin. Chronic infusion (2 mg/kg/day) resulted in significant increases in the velocity of circumferential shortening (baseline: 5.36+/-0.401; 14 days: 6.85+/-0.358 circ/s, p=0.049) and cardiac output (baseline: 0.142+/-0.019; 14 days: 0.25+/-0.019 l/min, p=0.001) as determined by 15 MHz echocardiography. Post-mortem corrected heart weights were not different between apelin and saline groups (p=0.5) and histology revealed no evidence of cellular hypertrophy in the apelin group (nuclei per unit area, p=0.9). Immunohistochemistry studies revealed APJ staining of myocardial cells in all regions of the adult mouse heart. Antibody staining, as well as quantitative real time polymerase chain reaction identified expression of both APJ and apelin in embryonic myocardium as early as embryonic day 13.5. CONCLUSIONS: Apelin reduces left ventricular preload and afterload and increases contractile reserve without evidence of hypertrophy. These results associate apelin with a positive hemodynamic profile and suggest it as an attractive target for pharmacotherapy in the setting of heart failure.

Protecting the myocardium: a role for the beta2 adrenergic receptor in the heart.

OBJECTIVE: The sympathetic nervous system enhances cardiac muscle function by activating beta adrenergic receptors (betaARs). Recent studies suggest that chronic betaAR stimulation is detrimental, however, and that it may play a role in the clinical deterioration of patients with congestive heart failure. To examine the impact of chronic beta1AR and beta2AR subtype stimulation individually, we studied the cardiovascular effects of catecholamine infusions in betaAR subtype knockout mice (beta1KO, beta2KO). DESIGN: Prospective, randomized, experimental study. SETTING: Animal research laboratory. SUBJECTS: beta1KO and beta2KO mice and wild-type controls. INTERVENTIONS: The animals were subjected to 2 wks of continuous infusion of the betaAR agonist isoproterenol. Analyses of cardiac function and structure were performed during and 3 days after completion of the infusions. Functional studies included graded exercise treadmill testing, in vivo assessments of left ventricular function using Mikro-Tip catheter transducers, right ventricular pressure measurements, and analyses of organ weight to body weight ratios. Structural studies included heart weight measurements, assessments of myocyte ultrastructure using electron microscopy, and in situ terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling staining to quantitate myocyte apoptosis. MEASUREMENTS AND MAIN RESULTS: We found that isoproterenol-treated beta2KO mice experienced greater mortality rates (p =.001, chi-square test using Fisher's exact method) and increased myocyte apoptosis at 3- and 7-day time points (p =.04 and p =.0007, respectively, two-way analysis of variance). CONCLUSION: The results of this study suggest that in vivo beta2AR activation is antiapoptotic and contributes to myocardial protection.