A State of Natriuretic Peptide Deficiency.

Measurement of natriuretic peptides (NPs) has proven its clinical value as biomarker, especially in the context of heart failure (HF). In contrast, a state of partial NP deficiency appears integral to several conditions in which lower NP concentrations in plasma presage overt cardiometabolic disease. Here, obesity and type 2 diabetes have attracted considerable attention. Other factors-including age, sex, race, genetics, and diurnal regulation-affect the NP "armory" and may leave some individuals more prone to development of cardiovascular disease. The molecular maturation of NPs has also proven complex, with highly variable O-glycosylation within the biosynthetic precursors. The relevance of this regulatory step in post-translational propeptide maturation has recently become recognized in biomarker measurement/interpretation and cardiovascular pathophysiology. An important proportion of people appear to have reduced effective net NP bioactivity in terms of receptor activation and physiological effects. The state of NP deficiency both entails a potential for further biomarker development and could also offer novel pharmacological possibilities. Alleviating the state of NP deficiency before development of overt cardiometabolic disease in selected patients could be a future path for improving precision medicine.

The counterregulatory response to hypoglycaemia in the pig.

The domestic pig is commonly used as animal model in the pharmaceutical development of new therapeutics for treatment of diabetes. Since a formal definition of hypoglycaemia only exists in humans, the purpose of this study was to assess the counterregulatory response in the domestic pig at glucose levels known to induce symptoms of hypoglycaemia in humans. Six pigs were included in hyperinsulinaemic glucose clamps with plasma glucose targets of 2, 3 and 5 mmol/L in a cross-over design, and the associated glucose counterregulatory response was assessed by measuring glucose kinetics and levels of glucagon, c-peptide, catecholamines, cortisol and growth hormone. Results showed that the 2 and 3 vs 5 mmol/L clamps significantly decreased and increased the secretion of c-peptide and glucagon, respectively (P < .05). This finding was associated with increased rate of glucose appearance (Ra ) and decreased rate of glucose disappearance (Rd ) (P < .001). No marked differences in the catecholamine, growth hormone or cortisol response were observed. Consequently, like humans, pigs respond to hypoglycaemia by decreasing the pancreatic output of insulin while increasing that of glucagon, with increased glucose mobilization and decreased glucose disposal as a result. The hypoglycaemic clamps did not result in a marked secretion of the other counterregulatory hormones.

Myocardial Changes in Diabetic and Nondiabetic Nonhuman Primates.

Diabetic human patients have increased risk of heart failure compared to healthy subjects. The underlying mechanisms for this are not fully understood, and to help develop improved treatment strategies, well-characterized animal models are essential. To investigate cardiac dysfunction in diabetes, this study evaluated myocardial changes in 10 aging rhesus monkeys with and without diabetes. Based on evaluation of plasma glycosylated hemoglobin and glucose, 7 of 10 rhesus macaques had diabetes for a minimum of 11 months, while 3 of 10 were categorized as nondiabetic. A detailed histological examination of formalin-fixed left ventricular myocardial samples was followed by a semiquantitative evaluation of myocardial fibrosis and fat infiltration; digital quantifications of myocardial collagen, lipofuscin, and nuclear area fractions; and measurements of cardiomyocyte diameter. Histological myocardial evaluation revealed the presence of lipofuscin; large nuclei; interstitial, replacement, and vascular fibrosis; adipocyte infiltration; and vacuolar degeneration with atrophy of cardiomyocytes and fibrosis. However, there were no differences between groups for semiquantitative fat infiltration, fibrosis, cardiomyocyte size, collagen, or nuclear and lipofuscin area fraction. Lipofuscin area fraction correlated with plasma insulin, triglyceride, total cholesterol, and high-density lipoprotein cholesterol concentrations. In conclusion, myocardial pathological changes were found in left ventricular myocardium in aged rhesus macaques, independent of the stage of diabetes. The duration of diabetes might have been too short to cause differences between groups.

18F-FDG PET/MR-imaging in a Göttingen Minipig model of atherosclerosis: Correlations with histology and quantitative gene expression.

BACKGROUND AND AIMS: The advantage of combining molecular and morphological imaging, e.g. positron emission tomography and magnetic resonance imaging (PET/MRI), is reflected in the increased use of these modalities as surrogate end-points in clinical trials. This study aimed at evaluating plaque inflammation using 18F-fluorodeoxyglucose (18F-FDG)-PET/MRI, and gene expression in a minipig model of atherosclerosis. METHODS: Göttingen Minipigs were fed for 60 weeks with fat/fructose/cholesterol-rich diet (FFC), chow (Control) or FFC-diet changed to chow midway (diet normalization group; DNO). In all groups, 18F-FDG-PET/MRI of the abdominal aorta was assessed midway and at study-end. The aorta was analyzed using histology and gene expression. RESULTS: At study-end, FFC had significantly higher FDG-uptake compared to Control (target-to-background maximal uptake, TBRMax (95% confidence interval) CITBRMax: 0.092; 7.32) and DNO showed significantly decreased uptake compared to FFC (CITBRMax: -5.94;-0.07). No difference was observed between DNO and Control (CITBRMax: -2.71; 4.11). FFC displayed increased atherosclerosis and gene expression of inflammatory markers, including vascular cell adhesion molecule 1 (VCAM-1), cluster of differentiation 68 (CD68), matrix metalloproteinase 9 (MMP9), cathepsin K (CTSK) and secreted phosphoprotein 1 (SPP1) compared to Control and DNO (all, p < 0.05). FDG-uptake correlated with gene expression of inflammatory markers, including CD68, ρs = 0.58; MMP9, ρs = 0.46; SPP1, ρs = 0.44 and CTSK, ρs = 0.49; (p ≤ 0.01 for all). CONCLUSIONS: In a model of atherosclerosis, 18F-FDG-PET/MRI technology allows for detection of inflammation in atherosclerotic plaques, consistent with increased inflammatory gene expression. Our findings corroborate clinical data and are important in pre-clinical drug development targeting plaque inflammation.

Role of age, Rho-kinase 2 expression, and G protein-mediated signaling in the myogenic response in mouse small mesenteric arteries.

The myogenic response (MR) and myogenic tone (MT) in resistance vessels is crucial for maintaining peripheral vascular resistance and blood flow autoregulation. Development of MT involves G protein-coupled receptors, and may be affected by aging. AIMS: (1) to estimate the mesenteric blood flow in myogenically active small mesenteric arteries; (2) to investigate the signaling from Gαq/11 and/or Gα12 activation to MT development; (3) to investigate the role of Rho-kinase 2 and aging on MT in mesenteric resistance arteries. METHODS: we used pressure myography, quantitative real-time PCR, and immunolocalization to study small (<200 µm) mesenteric arteries (SMA) from young, mature adult, and middle aged mice. RESULTS: Poiseuille flow calculations indicated autoregulation of blood flow at 60-120 mm Hg arterial pressure. Gαq/11 and Gα12 were abundantly expressed at the mRNA and protein levels in SMA. The Gαq/11 inhibitor YM-254890 suppressed MT development, and the Phosholipase C inhibitors U73122 and ET-18-OCH3 robustly inhibited it. We found an age-dependent increase in ROCK2 mRNA expression, and in basal MT. The specific ROCK2 inhibitor KD025 robustly inhibited MT in SMAs in all mice with an age-dependent variation in KD025 sensitivity. The inhibitory effect of KD025 was not prevented by the L-type Ca2+ channel activator BayK 8644. KD025 reversibly inhibited MT and endothelin-1 vasoconstriction in small pial arteries from Göttingen minipigs. CONCLUSIONS: MT development in SMAs occurs through a Gαq/11 /PLC/Ca2+ -dependent pathway, and is maintained via ROCK2-mediated Ca2+ sensitization. Increased MT at mature adulthood can be explained by increased ROCK2 expression/activity.

Age- and Sex-Associated Effects on Acute-Phase Proteins in Göttingen Minipigs.

Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP in Göttingen minipigs and to investigate the effects of age, sex, and various stimuli on these ranges. Serum concentrations of C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, pig major acute-phase protein (PMAP), albumin, and porcine α-1 acid glycoprotein (PAGP) were evaluated in 4 age groups (6, 16, 24 and 40-48 wk) of male and female Göttingen minipigs. In addition, minipigs were tested under 2 housing conditions, after acute LPS challenge, and after diet-induced obesity with and without mild diabetes. Changing the pigs to a new environment induced significant increases in CRP, PMAP, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only in obese pigs without diabetes. In conclusion, reference values for CRP, PMAP, haptoglobin, SAA, PAGP and albumin were established for male and female Göttingen minipigs of different ages. These APP were influenced by age and sex, underlining the importance of considering these factors when designing and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli.

Noninvasive assessment of pulse-wave velocity and flow-mediated vasodilation in anesthetized Göttingen minipigs.

Few methods for noninvasive assessment of arterial stiffness and endothelial dysfunction in porcine models are available. The aim of this study was to evaluate methods for assessment of arterial stiffness and endothelial dysfunction in anesthetized Göttingen minipigs. Pulse-wave velocity (PWV) was assessed in male Göttingen minipigs (n = 8; age approximately 60 wk) by using applanation tonometry of the carotid and femoral arteries. In addition, flow-mediated vasodilation (FMD) was assessed by using vascular ultrasonography of the brachial artery to evaluate endothelial dysfunction. To evaluate the reproducibility of the methods, minipigs were anesthetized by intravenous infusion of ketamine and midazolam and examined every other day for a total of 3 trials. Neither examination day nor systolic, diastolic, or mean arterial blood pressure statistically influenced PWV or FMD. The median interexamination coefficient of variation was 17% for PWV and 59% for FMD. Measured values of PWV corresponded largely to those in clinically healthy humans, but FMD values were lower than expected for lean, young animals. Although the ketamine-midazolam anesthesia we used has been associated with minor hemodynamic effects in vivo, in vitro studies suggest that both drugs are vasodilatory. Therefore anesthesia might have influenced the endothelial response, contributing to the modest FMD response and the concurrent high coefficients of variation that we noted. We conclude that PWV­but not FMD­showed acceptable interexamination variation for its potential application in porcine models.

Feasibility of simultaneous PET/MR in diet-induced atherosclerotic minipig: a pilot study for translational imaging.

Novel hybrid 18-fluoro-deoxy-D-glucose ((18)F-FDG) based positron emission tomography (PET) and magnetic resonance imaging (MRI) has shown promise for characterization of atherosclerotic plaques clinically. The purpose of this study was to evaluate the method in a pre-clinical model of diet-induced atherosclerosis, based on the Göttingen minipig. Using (18)F-FDG PET/MRI the goal was to develop and create a new imaging method in an in vivo animal model for translational studies of atherosclerosis. We used a strategy of multisequence MRI for optimal anatomical imaging of the abdominal aortas of the pigs (n=4): T1-weighted turbo spin-echo (T1-TSE), T2-weighted turbo spin-echo (T2-TSE) and proton density imaging with and without fat saturation. (18)F-FDG PET emission data were collected from a single bed position of the abdominal aorta in 3D mode for either 10 (n=4) or 10 and 20 minutes (n=2) to measure glycolysis as given by standardized uptake values (SUV). Ex vivo en face evaluation of aortas from an atherosclerotic animal illustrated plaque distribution macroscopically, compared to a lean control animal. Although T2-TSE weighted imaging was most consistent, no one MRI sequence was preferable and superior to another for visualization and identification of the abdominal aorta. We found poor correlation between SUVs obtained from 10 and 20 minutes of reconstructed PET emission data. This can most likely be ascribed to intestinal movement. In conclusion multisequence MRI is recommended for optimal imaging of the abdominal aorta using MRI. Furthermore we found that 10 minutes of PET emission data seems adequate. This is the first study to demonstrate that the method of (18)F-FDG PET/MRI is feasible in minipig models of atherosclerosis, and therefore relevant in larger prospective studies. Perspectives of the method include correlation to e.g. aortic immunohistochemistry findings and a range of genomic and proteomic analyses.