GIP_HUMAN[22–51] is a new proatherogenic peptide identified by native plasma peptidomics

We recently established a new plasma peptidomic technique and comprehensively identified a large number of low-molecular weight and low-abundance native peptides using a single drop of human plasma. To discover a novel polypeptide that potently modulates the cardiovascular system, we performed a bioinformatics analysis of the large-scale identification results, sequentially synthesized the selected peptide sequences, tested their biological activities, and identified a 30-amino-acid proatherogenic peptide, GIP_HUMAN[22–51], as a potent proatherosclerotic peptide hormone. GIP_HUMAN[22–51] has a common precursor with the glucose-dependent insulinotropic polypeptide (GIP) and is located immediately N-terminal to GIP. Chronic infusion of GIP_HUMAN[22–51] into ApoE−/− mice accelerated the development of aortic atherosclerotic lesions, which were inhibited by co-infusions with an anti-GIP_HUMAN[22–51] antibody. GIP_HUMAN[22–51] increased the serum concentrations of many inflammatory and proatherogenic proteins, whereas neutralising antibodies reduced their levels. GIP_HUMAN[22–51] induced IκB-α degradation and nuclear translocation of NF-κB in human vascular endothelial cells and macrophages. Immunoreactive GIP_HUMAN[22–51] was detected in human tissues but there was no colocalization with the GIP. The plasma GIP_HUMAN[22–51] concentration in healthy humans determined using a stable-isotope tagged peptide was approximately 0.6 nM. This study discovered a novel endogenous proatherogenic peptide by using a human plasma native peptidomic resource.

Human and preclinical studies of the host–gut microbiome co-metabolite hippurate as a marker and mediator of metabolic health

ObjectiveGut microbial products are involved in regulation of host metabolism. In human and experimental studies, we explored the potential role of hippurate, a hepatic phase 2 conjugation product of microbial benzoate, as a marker and mediator of metabolic health.DesignIn 271 middle-aged non-diabetic Danish individuals, who were stratified on habitual dietary intake, we applied 1H-nuclear magnetic resonance (NMR) spectroscopy of urine samples and shotgun-sequencing-based metagenomics of the gut microbiome to explore links between the urine level of hippurate, measures of the gut microbiome, dietary fat and markers of metabolic health. In mechanistic experiments with chronic subcutaneous infusion of hippurate to high-fat-diet-fed obese mice, we tested for causality between hippurate and metabolic phenotypes.ResultsIn the human study, we showed that urine hippurate positively associates with microbial gene richness and functional modules for microbial benzoate biosynthetic pathways, one of which is less prevalent in the Bacteroides 2 enterotype compared with Ruminococcaceae or Prevotella enterotypes. Through dietary stratification, we identify a subset of study participants consuming a diet rich in saturated fat in which urine hippurate concentration, independently of gene richness, accounts for links with metabolic health. In the high-fat-fed mice experiments, we demonstrate causality through chronic infusion of hippurate (20 nmol/day) resulting in improved glucose tolerance and enhanced insulin secretion.ConclusionOur human and experimental studies show that a high urine hippurate concentration is a general marker of metabolic health, and in the context of obesity induced by high-fat diets, hippurate contributes to metabolic improvements, highlighting its potential as a mediator of metabolic health.

β-arrestin-mediated Angiotensin II type 1 Receptor Activation Promotes Pulmonary Vascular Remodeling in Pulmonary Hypertension

ObjectivesThe goal of this study was to test whether a β-arrestin-biased agonist of the angiotensin II (AngII) type 1 receptor (AT1R), which acts as a vasodilator while not blocking cellular proliferation, would have positive effects compared to a balanced agonist, angiotensin II (AngII), or an antagonist, losartan, in pulmonary arterial hypertension (PAH).BackgroundPAH is a disease of abnormal pulmonary vascular remodeling whose treatment has focused on targeting vasoactive substances, such as inhibiting endothelin signaling and promoting prostacyclin signaling. PAH medical therapies are thought to primarily act as vasodilators, although they may also have effects on pulmonary vascular remodeling. There are a number of reports that blocking AT1R signaling can be beneficial in preclinical models of PAH. The AT1R is a G protein-coupled receptor (GPCR) that promotes vasoconstriction through heterotrimeric G proteins but also signals via β-arrestins, which promote cardioprotective effects and vasodilation.MethodsWe compared the effects of a β-arrestin-biased AT1R agonist, TRV120023 (TRV023), to a balanced agonist (AngII) and an antagonist (losartan) in preclinical PAH models.ResultsIn acute infusion studies, AngII increased right ventricular (RV) pressures while TRV023 did not. However, with chronic infusion in monocrotaline (MCT) PAH rats, TRV023 failed to improve hemodynamics or survival compared to AngII, while losartan significantly improved survival. Both TRV023 and AngII enhanced proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) from PAH patients, which was associated with the promotion of proliferative MAP kinase signaling.Conclusionsβ-arrestin-mediated AT1R signaling promotes vascular remodeling and worsens PAH, and suggests that the primary benefit of current PAH therapies is through pulmonary vascular reverse remodeling and not vasodilation.

Chronically infused angiotensin II induces depressive-like behavior via microglia activation

Brain inflammation is one of hypotheses explaining complex pathomechanisms of depression. Angiotensin II (ANGII), which is associated with hypertension, also induces brain inflammation. However, there is no animal study showing the direct relationship between ANGII and depression. To address this issue, ANGII-containing osmotic pumps were implanted into adult male C57BL/6 mice subcutaneously for subacute (7 days) and chronic (at least 21 days) periods and behavioral and molecular analyses were conducted. Chronic infusion of ANGII into mice induced depressive-like behaviors, including the tail suspension test and forced swimming test, which were reversed by imipramine. Chronic infusion of ANGII also induced microglial activation in the hippocampus with increase of Il-1β mRNA and decrease of Arg1 mRNA. In addition, chronic ANGII infusion activated the hypothalamic–pituitary–adrenal axis (HPA axis) and resulted in decreased hippocampal glucocorticoid receptor level. However, subacute ANGII infusion did not induce significant molecular and behavioral changes in mice compared to that of control. The molecular and behavioral changes by chronic ANGII infusion were reversed by co-treatment of minocycline or telmisartan. In addition, ANGII treatment also induced the pro-inflammatory changes in BV-2 microglial cells. Our results indicate that ANGII can induce depressive-like behaviors via microglial activation in the hippocampus and HPA axis hyperactivation in mice. These might suggest possible mechanism on depressive symptom in chronic hypertensive state.

IL-10 treatment decreases blood pressure in male, but not female, spontaneously hypertensive rats

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that induces nitric oxide (NO) production. IL-10 supplementation has been previously shown to lower blood pressure (BP) in male hypertensive mice, but the effect of exogenous IL-10 in hypertensive female rodents has not been studied. For the present study, we hypothesized that chronic infusion of IL-10 in hypertensive rats would lower BP concomitant with an increase in renal NO synthase (NOS) activity. Male and female spontaneously hypertensive rats (SHRs; 12 wk old) were randomized to receive IL-10 infusion by subcutaneous minipump (3.5 µg·kg−1·day−1) or serve as sham controls ( n = 4–6 rats per treatment per sex). BP was measured by tail cuff before and after 2 wk of treatment. Renal T cells and IL-10 were measured by flow cytometry, and NOS activity was determined by conversion of radiolabeled arginine to radiolabeled citrulline. Female SHRs had greater IL-10+ renal cells than male SHRs and greater expression of the IL-10 receptor at baseline. BP did not change in female SHRs treated with IL-10, but BP significantly decreased following IL-10 infusion in male SHRs. Contrary to our hypothesis, NOS enzymatic activity decreased with IL-10 treatment in the renal inner medulla and cortex of both sexes. Renal regulatory T cells also decreased in both sexes after IL-10 treatment. In conclusion, despite male SHRs having less IL-10 and IL-10 receptor expression in the kidney compared with female SHRs, exogenous IL-10 selectively decreased BP only in male SHRs. Furthermore, our data suggest that exogenous IL-10-induced decreases in BP in male SHRs are not dependent on upregulating renal NOS activity.

Validation of an effective implantable pump-infusion system for chronic convection-enhanced delivery of intracerebral topotecan in a large animal model

OBJECTIVEIntracerebral convection-enhanced delivery (CED) has been limited to short durations due to a reliance on externalized catheters. Preclinical studies investigating topotecan (TPT) CED for glioma have suggested that prolonged infusion improves survival. Internalized pump-catheter systems may facilitate chronic infusion. The authors describe the safety and utility of long-term TPT CED in a porcine model and correlation of drug distribution through coinfusion of gadolinium.METHODSFully internalized CED pump-catheter systems were implanted in 12 pigs. Infusion algorithms featuring variable infusion schedules, flow rates, and concentrations of a mixture of TPT and gadolinium were characterized over increasing intervals from 4 to 32 days. Therapy distribution was measured using gadolinium signal on MRI as a surrogate. A 9-point neurobehavioral scale (NBS) was used to identify side effects.RESULTSAll animals tolerated infusion without serious adverse events. The average NBS score was 8.99. The average maximum volume of distribution (Vdmax) in chronically infused animals was 11.30 mL and represented 32.73% of the ipsilateral cerebral hemispheric volume. Vdmax was achieved early during infusions and remained relatively stable despite a slight decline as the infusion reached steady state. Novel tissue TPT concentrations measured by liquid chromatography mass spectroscopy correlated with gadolinium signal intensity on MRI (p = 0.0078).CONCLUSIONSProlonged TPT-gadolinium CED via an internalized system is safe and well tolerated and can achieve a large Vdmax, as well as maintain a stable Vd for up to 32 days. Gadolinium provides an identifiable surrogate for measuring drug distribution. Extended CED is potentially a broadly applicable and safe therapeutic option in select patients.

Evaluating infusate parameters for direct drug delivery to the brainstem: a comparative study of convection-enhanced delivery versus osmotic pump delivery

OBJECTIVEConvection-enhanced delivery (CED) and osmotic pump delivery both have been promoted as promising techniques to deliver drugs to pediatric diffuse intrinsic pontine gliomas (DIPGs). Correspondingly, the aim of this study was to understand how infusate molecular weight (MW), duration of delivery, and mechanism of delivery (CED or osmotic pump) affect volume of distribution (Vd) in the brainstem, to better inform drug selection and delivery in future DIPG investigations.METHODSA series of in vivo experiments were conducted using rat models. CED and osmotic pump delivery systems were surgically implanted in the brainstem, and different MW fluorescent dextran beads were infused either once (acute) or daily for 5 days (chronic) in a volume infused (Vi). Brainstems were harvested after the last infusion, and Vd was quantified using serial sectioning and fluorescence imaging.RESULTSFluorescence imaging showed infusate uptake within the brainstem for both systems without complication. A significant inverse relationship was observed between infusate MW and Vd in all settings, which was distinctly exponential in nature in the setting of acute delivery across the 570-Da to 150-kDa range. Chronic duration and CED technique resulted in significantly greater Vd compared to acute duration or osmotic pump delivery, respectively. When accounting for Vi, acute infusion yielded significantly greater Vd/Vi than chronic infusion. The distribution in CED versus osmotic pump delivery was significantly affected by infusate MW at higher weights.CONCLUSIONSHere the authors demonstrate that infusate MW, duration of infusion, and infusion mechanism all impact the Vd of an infused agent and should be considered when selecting drugs and infusion parameters for novel investigations to treat DIPGs.

Down-regulation of Brain Gα12 attenuates Angiotensin II Dependent Hypertension

Background Angiotensin II (Ang II) activates central Angiotensin II type 1 receptors (AT1R) to increase blood pressure (BP) via multiple pathways. However, whether central Gα proteins contribute to Ang II induced hypertension remains unknown. We hypothesized that AT1R couple with Gα12 and/or Gαq to produce sympatho-excitation and increase BP and downregulation of these Gα subunit proteins will attenuate Ang II dependent hypertension. Methods & Results After chronic infusion of Ang II (s.c. 350 ng/kg/min) or vehicle for 2-weeks, Ang II evoked an increase in Gα12 expression, but not Gαq in the RVLM of Sprague-Dawley rats. In other studies, rats that received Ang II or vehicle infusion s.c. were simultaneously infused i.c.v. with a scrambled (SCR) or Gα12 oligodeoxynucleotide (ODN; 50 μg/day). Central Gα12 ODN infusion lowered mean BP in Ang II infused rats compared with SCR ODN infusion (14- day peak; 133 ± 12 vs 176 ± 11 mmHg). Compared to the SCR ODN group, Ang II infused rats that received i.c.v. Gα12 ODN showed a greater increase in heart rate to atropine, an attenuated reduction in BP to chlorisondamine, and an improved baroreflex sensitivity. In addition, central Gα12 and Gαq ODN pretreatment blunted the pressor response to an acute i.c.v. injection of Ang II (i.c.v., 200 ng). Conclusions These findings suggest that central Gα12 protein signaling pathways play an important role in the development of chronic AngII-dependent hypertension in rats.