Nanopore sequencing reveals methylation changes associated with obesity in circulating cell-free DNA from Göttingen Minipigs.

Profiling of circulating cell-free DNA (cfDNA) by tissue-specific base modifications, such as 5-methylcytosines (5mC), may enable the monitoring of ongoing pathophysiological processes. Nanopore sequencing allows genome-wide 5mC detection in cfDNA without bisulphite conversion. The aims of this study were: i) to find differentially methylated regions (DMRs) of cfDNA associated with obesity in Göttingen minipigs using Nanopore sequencing, ii) to validate a subset of the DMRs using methylation-specific PCR (MSP-PCR), and iii) to compare the cfDNA DMRs with those from whole blood genomic DNA (gDNA). Serum cfDNA and gDNA were obtained from 10 lean and 7 obese Göttingen Minipigs both with experimentally induced myocardial infarction and sequenced using Oxford Nanopore MinION. A total of 1,236 cfDNA DMRs (FDR<0.01) were associated with obesity. In silico analysis showed enrichment of the adipocytokine signalling, glucagon signalling, and cellular glucose homoeostasis pathways. A strong cfDNA DMR was discovered in PPARGC1B, a gene linked to obesity and type 2 diabetes. The DMR was validated using MSP-PCR and correlated significantly with body weight (P < 0.05). No DMRs intersected between cfDNA and gDNA, suggesting that cfDNA originates from body-wide shedding of DNA. In conclusion, nanopore sequencing detected differential methylation in minute quantities (0.1-1 ng/µl) of cfDNA. Future work should focus on translation into human and comparing 5mC from somatic tissues to pinpoint the exact location of pathology.

Oxford nanopore sequencing can reveal changes in methylation patterns associated with obesity in minute quantities of cell-free DNA from serum.Bisulphite conversion and methylation-specific PCR can be used to validate differentially methylated regions in cell-free DNA.A differentially methylated region in an intronic region of the PPARGC1B gene was found associated with obesity.Differentially methylated regions in cell-free DNA could be useful as early risk markers of certain diseases and pathologies.

Functional and morphological renal changes in a Göttingen Minipig model of obesity-related and diabetic nephropathy.

Obesity-related glomerulopathy and diabetic nephropathy (DN) are serious complications to metabolic syndrome and diabetes. The purpose was to study effects of a fat, fructose and cholesterol-rich (FFC) diet with and without salt in order to induce hypertension on kidney function and morphology in Göttingen Minipigs with and without diabetes. Male Göttingen Minipigs were divided into 4 groups: SD (standard diet, n = 8), FFC (FFC diet, n = 16), FFC-DIA (FFC diet + diabetes, n = 14), FFC-DIA + S (FFC diet with extra salt + diabetes, n = 14). Blood and urine biomarkers, glomerular filtration rate (GFR), blood pressure (BP) and resistive index (RI) were evaluated after 6-7 months (T1) and 12-13 months (T2). Histology, electron microscopy and gene expression (excluding FFC-DIA + S) were evaluated at T2. All groups fed FFC-diet displayed obesity, increased GFR and RI, glomerulomegaly, mesangial expansion (ME) and glomerular basement membrane (GBM) thickening. Diabetes on top of FFC diet led to increased plasma glucose and urea and proteinuria and tended to exacerbate the glomerulomegaly, ME and GBM thickening. Four genes (CDKN1A, NPHS2, ACE, SLC2A1) were significantly deregulated in FFC and/or FFC-DIA compared to SD. No effects on BP were observed. Göttingen Minipigs fed FFC diet displayed some of the renal early changes seen in human obesity. Presence of diabetes on top of FFC diet exacerbated the findings and lead to changes resembling the early phases of human DN.

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.

Optimization of pig models for translation of subcutaneous pharmacokinetics of therapeutic proteins: Liraglutide, insulin aspart and insulin detemir.

Prediction of human pharmacokinetics (PK) from data obtained in animal studies is essential in drug development. Here, we present a thorough examination of how to achieve good pharmacokinetic data from the pig model for translational purposes by using single-species allometric scaling for selected therapeutic proteins: liraglutide, insulin aspart and insulin detemir. The predictions were based on non-compartmental analysis of intravenous and subcutaneous PK data obtained from two injection regions (neck, thigh) in two pig breeds, domestic pig and Göttingen Minipig, that were compared with PK parameters reported in humans. The effects of pig breed, injection site and injection depth (insulin aspart only) on the PK of these proteins were also assessed. Results show that the prediction error for human PK was within two-fold for most PK parameters in both pig breeds. Furthermore, pig breed significantly influenced the plasma half-life and mean absorption time (MAT), both being longer in Göttingen Minipigs compared to domestic pigs (P <0.01). In both breeds, thigh vs neck dosing was associated with a higher dose-normalized maximum plasma concentration and area under the curve as well as shorter MAT and plasma half-life (P <0.01). Finally, more superficial injections resulted in faster absorption, higher Cmax/dose and bioavailability of insulin aspart (P <0.05, 3.0 vs 5.0 mm injection depth). In conclusion, pig breed and injection region affected the PK of liraglutide, insulin aspart and insulin detemir and reliable predictions of human PK were demonstrated when applying single-species allometric scaling with the pig as a pre-clinical animal model.

Is there a correlation between functional results and radiographic findings in patients with distal radius fracture A0 type A3 treated with volar locking plate or external fixator?

The aim of this study was to test the hypothesis that precise restoration of distal radius fractures is correlated to better patient-reported outcome. METHODS: The correlation between radiographic results and functional outcome was explored in 156 patients with extra-articular distal radius fractures included in a multicenter, randomized controlled trial comparing 2 surgical interventions, Volar Locking Plate or External Fixator. The primary functional outcome was the Patient Rated Wrist and Hand Evaluation score (PRWHE). Radiographically we assessed volar tilt, radial inclination, radial height, ulnar variance, and the presence of ulnar styloid fracture. The Pearson correlation analysis was used to estimate correlations between parameters. RESULTS: At 1-year follow-up the mean difference in radiographic findings compared with the uninjured side (min, max) was: reduced volar tilt 5.3° (-15°, 25°), reduced radial inclination 2.3° (-6°, 12°), radial height 1.3 mm (-4 mm, 7 mm), and ulnar variance -0.5 mm (-6 mm, 3 mm). Overall, we found no correlation between radiographic parameters and the PRWHE at 1-year follow-up within the whole group, regardless of which treatment was chosen. At the time of injury 53% (N = 80) had sustained an additional ulnar styloid fracture. After 1 year this fracture was still radiographically present in 31% (N = 43) of the patients. No correlation between PRWHE score and the presence of an ulnar styloid fracture at 1-year follow-up was found. CONCLUSIONS: We found no correlation between functional outcome (PRWHE) and radiographic findings after 1 year in patients operated on with a Volar Locking Plate or External Fixator. Patient-specific factors were more important than radiographic measurements in this study group.Level of evidence: Therapeutic Level 2Trial registration: Norway: National Committee for Medical and Health Research Ethics 213/555ClinicalTrials.gov ID: NCT01904084Randomization of first patient: 02.09.2013.

[68Ga]Ga-NODAGA-E[(cRGDyK)]2 Angiogenesis PET/MR in a Porcine Model of Chronic Myocardial Infarction.

Angiogenesis is crucial in tissue repair and prevents scar tissue formation following an ischemic event such as myocardial infarction. The ischemia induces formation of new capillaries, which have high expression of integrin αvß3. [68Ga]Ga-NODAGA-E[(cRGDyK)]2 ([68Ga]Ga-RGD) is a promising PET-radiotracer reflecting angiogenesis by binding to integrin αvß3. A Göttingen mini-pig underwent transient catheter-induced left anterior descending artery (LAD) occlusion for 120 min, and after 8 weeks was imaged on a Siemens mMR 3T PET/MR system. A large antero-septal infarction was evident by late gadolinium enhancement (LGE) on the short axis and 2-4 chamber views. The infarcted area corresponded to the area with high [68Ga]Ga-RGD uptake on the fused PET/MR images, with no uptake in the healthy myocardium. To support the hypothesis that [68Ga]Ga-RGD uptake reflects angiogenesis, biopsies were sampled from the infarct border and healthy myocardium. Expression of αvß3 was evaluated using immunohistochemistry. The staining showed higher αvß3 expression in the capillaries of the infarct border compared to those in the healthy myocardium. These initial data confirm in vivo detection of angiogenesis using [68Ga]Ga-RGD PET in a translational model, which overall support the method applicability when evaluating novel cardio-protective therapies.

Atorvastatin impairs liver mitochondrial function in obese Göttingen Minipigs but heart and skeletal muscle are not affected.

Statins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.

Surgical Treatment of Distal Radial Fractures with External Fixation Versus Volar Locking Plate: A Multicenter Randomized Controlled Trial.

BACKGROUND: The use of volar locking plate fixation (VLP) for unstable extra-articular distal radial fractures has increased in the last decades. External fixation (EF) is less frequently used. This change of surgical approach has only to some extent been evidence-based. METHODS: In this multicenter, randomized controlled trial, we compared VLP and EF in patients between 18 and 70 years of age who had a displaced extra-articular distal radial fracture (OTA/AO type A3). The patients were examined at 6 weeks, 3 months, and 1 year postoperatively. The primary outcome measure was the Patient-Rated Wrist/Hand Evaluation score (PRWHE). Secondary outcomes were the shortened version of the Disabilities of the Arm, Shoulder and Hand (QuickDASH), pain score on a visual analog scale (VAS), and radiographic measurements. Range of motion, grip strength, finger stiffness, complications, and reoperations were also recorded. RESULTS: One hundred and fifty-six patients were included. One hundred and forty-two (91%)-127 women (89%) and 15 men (11%)-completed 1 year of follow-up. Sixty-nine patients were treated with VLP and 73, with EF. The mean age was 56 years. At 6 weeks, the median PRWHE score was significantly higher in the EF group (44) compared with the VLP group (27) (p < 0.001). At 3 months and 1 year, the difference between groups was not significant. The median QuickDASH score was 27 in the VLP group and 43 in the EF group at 6 weeks (p < 0.001), and a significant difference persisted at 3 months (p = 0.023). The VLP group had superior results in terms pain during activity, wrist extension, and ulnar and radial deviation at 1 year, whereas the number of major complications was similar in the 2 groups. CONCLUSIONS: Patients treated with VLP had earlier recovery of function compared with patients treated with EF. One year postoperatively, we found no significant functional difference. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

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.

The expression signatures in liver and adipose tissue from obese Göttingen Minipigs reveal a predisposition for healthy fat accumulation.

BACKGROUND: Model animals are valuable resources for dissecting basic aspects of the regulation of obesity and metabolism. The translatability of results relies on understanding comparative aspects of molecular pathophysiology. Several studies have shown that despite the presence of overt obesity and dyslipidemia in the pig key human pathological hepatic findings such as hepatocellular ballooning and abundant steatosis are lacking in the model. OBJECTIVES: The aim of this study was to elucidate why these histopathological characteristics did not occur in a high fat, fructose and cholesterol (FFC) diet-induced obese Göttingen Minipig model. METHODS: High-throughput expression profiling of more than 90 metabolically relevant genes was performed in liver, subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) of male minipigs diet fed: standard chow (SD, n = 7); FFC diet (n = 14); FFC diet in streptozotocin-induced diabetic pigs (FFCDIA, n = 8). Moreover, histopathological assessment of SAT and VAT was performed. RESULTS: 12, 4 and 1 genes were highly significantly differentially expressed in liver, SAT and VAT when comparing the FFC and SD groups whereas the corresponding numbers were 15, 2, and 1 when comparing the FFCDIA and SD groups. Although the minipigs in both FFC groups developed sever obesity and dyslipidemia, the insulin-signaling pathways were not affected. Notably, four genes involved in lipid acquisition and removal, were highly deregulated in the liver: PPARG, LPL, CD36 and FABP4. These genes have been reported to play a major role in promoting hepatic steatosis in rodents and humans. Since very little macrophage-associated pro-inflammatory response was detected in the adipose tissues the expansion appears to have no adverse impact on adipose tissue metabolism. CONCLUSION: The study shows that morbidly obese Göttingen Minipigs are protected against many of the metabolic and hepatic abnormalities associated with obesity due to a remarkable ability to expand the adipose compartments to accommodate excess calories.

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.

Hyperglycemia-induced transcriptional regulation of ROCK1 and TGM2 expression is involved in small artery remodeling in obese diabetic Göttingen Minipigs.

Obesity and diabetes in humans are associated with hypertrophic remodeling and increased media:lumen ratio of small resistance arteries, which is an independent predictor of cardiovascular events. In order to minimize increases in media:lumen ratio, hypertrophic remodeling should be accompanied by outward remodeling. We aimed to investigate the mechanisms of structural remodeling in small pial arteries (PAs) and terminal mesenteric arteries (TMAs) from obese Göttingen Minipigs with or without diabetes. Göttingen Minipigs received either control diet (lean control (LC)), high fat/high fructose/high cholesterol diet (FFC), or FFC diet with streptozotocin (STZ)-induced diabetes (FFC/STZ) for 13 months. At the end of the study (20 months), we assessed body weight, fasting plasma biochemistry, passive vessel dimensions, mRNA expression (matrix metallopeptidases 2/9 (MMP2, MMP9), tissue inhibitor of metallopeptidase 1 (TIMP1), transglutaminase 2 (TGM2), Rho-kinase 1 (ROCK1), TGFß-receptor 2 (TGFBR2), and IGF1-receptor (IGFR1) genes), and immunofluorescence in PAs and TMAs. We performed multiple linear correlation analyses using plasma values, structural data, and gene expression data. We detected outward hypertrophic remodeling in TMAs and hypertrophic remodeling in PAs from FFC/STZ animals. ROCK1 and TGM2 genes were up-regulated in PAs and TMAs from the FFC/STZ group. Passive lumen diameter (PLD) of TMAs was correlated with plasma values of glucose (GLU), fructosamine (FRA), total cholesterol (TC), and triglycerides (TGs). ROCK1 and TGM2 expressions in TMAs were correlated with PLD, plasma GLU, fructosamine, and TC. ROCK1 and TGM2 proteins were immunolocalized in the media of PAs and TMAs, and their fluorescence levels were increased in the FFC/STZ group. Hyperglycemia/hyperlipidemia is involved in regulation of ROCK1 and TGM2 expression leading to outward remodeling of small resistance arteries in obese diabetic Göttingen Minipigs.

Experimental non-alcoholic steatohepatitis in Göttingen Minipigs: consequences of high fat-fructose-cholesterol diet and diabetes.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in humans, and ranges from steatosis to non-alcoholic steatohepatitis (NASH), the latter with risk of progression to cirrhosis. The Göttingen Minipig has been used in studies of obesity and diabetes, but liver changes have not been described. The aim of this study was to characterize hepatic changes in Göttingen Minipigs with or without diabetes, fed a diet high in fat, fructose, and cholesterol to see if liver alterations resemble features of human NAFLD/NASH. METHODS: Fifty-four male castrated minipigs (age 6 to 7 months) were distributed into four groups and diet-fed for 13 months. Groups were: lean controls fed standard diet (SD, n = 8), a group fed high fat/fructose/cholesterol diet (FFC, n = 16), a group fed high fat/fructose/cholesterol diet but changed to standard diet after 7 months (diet normalization, FFC/SD, n = 16), and a streptozotocin-induced diabetic group fed high fat/fructose/cholesterol diet (FFCDIA, n = 14). At termination, blood samples for analyses of circulating biomarkers and liver tissue for histopathological assessment and analyses of lipids and glycogen content were collected. RESULTS: In comparison with SD and FFC/SD, FFC and FFCDIA pigs developed hepatomegaly with increased content of cholesterol, whereas no difference in triglyceride content was found. FFC and FFCDIA groups had increased values of circulating total cholesterol and triglycerides and the hepatic circulating markers alkaline phosphatase and glutamate dehydrogenase. In the histopathological evaluation, fibrosis (mainly located periportally) and inflammation along with cytoplasmic alterations (characterized by hepatocytes with pale, granulated cytoplasm) were found in FFC and FFCDIA groups compared to SD and FFC/SD. Interestingly, FFC/SD also had fibrosis, a feature not seen in SD. Only two FFC and three FFCDIA pigs had > 5% steatosis, and no hepatocellular ballooning or Mallory-Denk bodies were found in any of the pigs. CONCLUSIONS: Fibrosis, inflammation and cytoplasmic alterations were characteristic features in the livers of FCC and FFCDIA pigs. Overall, diabetes did not exacerbate the hepatic changes compared to FFC. The limited presence of the key human-relevant pathological hepatic findings of steatosis and hepatocellular ballooning and the variation in the model, limits its use in preclinical research without further optimisation.

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.

Dietary normalization from a fat, fructose and cholesterol-rich diet to chow limits the amount of myocardial collagen in a Göttingen Minipig model of obesity.

BACKGROUND: Dietary interventions have been shown to attenuate some of the myocardial pathological alterations associated with obesity. This study evaluated the effect of dietary normalization from a fat/fructose/cholesterol-rich diet to chow on left ventricular (LV) myocardial fibrosis, fat infiltration and hypertrophy but also the specific influence of obesity, plasma lipids and glucose metabolism markers on heart morphology in a Göttingen Minipig model of obesity. METHODS: Forty castrated male Göttingen Minipigs were assigned to three groups fed either standard minipig chow (SD, n = 8) for 13 months, fat/fructose/cholesterol-rich diet (FFC, n = 16) for 13 months or fat/fructose/cholesterol-rich diet for 7 months and then changed to standard minipig chow for the remaining 6 months (FFC/SD, n = 16). Body weight, body fat percentage, plasma lipids and glucose metabolism markers were evaluated in all three groups after 6-7 months (prior to diet adjustment for FFC/SD) and again before termination. Further, biochemical quantification of myocardial collagen and triglyceride content, semi-quantitative histological evaluation of fibrosis and fat infiltration and quantitative histological analysis of collagen and cardiomyocyte diameter were performed and heart weight was obtained after termination. Group differences were evaluated using Kruskal-Wallis test and Fisher's exact test for categorical variables. Pearson correlation analysis was performed to test for correlations between myocardial changes and selected explanatory variables. For non-parametric response variables, a Spearman correlation analysis was applied. RESULTS: Myocardial collagen content quantified biochemically was significantly lower in FFC/SD compared to FFC (P = 0.02). Furthermore, dietary normalization from a fat/fructose/cholesterol-rich diet to chow caused stagnation of body weight and body fat percentage, normalized intravenous glucose tolerance index (KG) and plasma lipid levels. CONCLUSION: Dietary normalization led to lower LV collagen content in obese Göttingen Minipigs. Despite gross obesity and significant deteriorations in glucose and lipid metabolism, only mild myocardial changes were found in this model of obesity and therefore further model optimization is warranted in order to induce more severe myocardial changes before dietary or pharmacological interventions.

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.

Göttingen minipig model of diet-induced atherosclerosis: influence of mild streptozotocin-induced diabetes on lesion severity and markers of inflammation evaluated in obese, obese and diabetic, and lean control animals.

BACKGROUND: From a pharmacological perspective, readily-available, well-characterized animal models of cardiovascular disease, including relevant in vivo markers of atherosclerosis are important for evaluation of novel drug candidates. Furthermore, considering the impact of diabetes mellitus on atherosclerosis in human patients, inclusion of this disease aspect in the characterization of a such model, is highly relevant. The objective of this study was to evaluate the effect of mild streptozotocin-induced diabetes on ex- and in vivo end-points in a diet-induced atherosclerotic minipig model. METHODS: Castrated male Göttingen minipigs were fed standard chow (CD), atherogenic diet alone (HFD) or with superimposed mild streptozotocin-induced diabetes (HFD-D). Circulating markers of inflammation (C-reactive protein (CRP), oxidized low-density lipoprotein (oxLDL), plasminogen activator inhibitor-1, lipid and glucose metabolism were evaluated together with coronary and aortic atherosclerosis after 22 or 43 diet-weeks. Group differences were evaluated by analysis of variance for parametric data and Kruskal-Wallis test for non-parametric data. For qualitative assessments, Fisher's exact test was applied. For all analyses, p < 0.05 was considered statistically significant. RESULTS: Overall, HFD and HFD-D displayed increased CRP, oxLDL and lipid parameters compared to CD at both time points. HFD-D displayed impaired glucose metabolism as compared to HFD and CD. Advanced atherosclerotic lesions were observed in both coronary arteries and aorta of HFD and HFD-D, with more advanced plaque findings in the aorta but without differences in lesion severity or distribution between HFD and HFD-D. Statistically, triglyceride was positively (p = 0.0039), and high-density lipoprotein negatively (p = 0.0461) associated with aortic plaque area. CONCLUSIONS: In this model, advanced coronary and aortic atherosclerosis was observed, with increased levels of inflammatory markers, clinically relevant to atherosclerosis. No effect of mild streptozotocin-induced diabetes was observed on plaque area, lesion severity or inflammatory markers.

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.