Therapeutic Antibody Against Phosphorylcholine Preserves Coronary Function and Attenuates Vascular 18F-FDG Uptake in Atherosclerotic Mice.

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of 18F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.

Clearance of cardiac troponin T with and without kidney function.

OBJECTIVE: The extent of kidney-dependent clearance of the cardiac damage biomarker cardiac troponin T (cTnT) is not known. METHODS AND RESULTS: We examined clearance of cTnT after injection of heart extracts in rats with or without clamped kidney vessels. The extent of degradation of cTnT to fragments able to pass the glomerular membrane and the kidney extraction index of cTnT was examined in human subjects. After a bolus injection of rat cardiac extract, simulating a large myocardial infarction, there was no significant difference in clearance of cTnT with or without kidney function. However, a slower clearance was observed late in the clearance process, when cTnT levels were low. When low levels of rat cardiac extract were infused at a constant rate to steady state, clamping of the renal vessels resulted in significant 2-fold reduction in clearance of cTnT. Over 60% of the measured cTnT in human subjects had a molecular weight below 17kDa, expected to have a relatively free passage over the glomerular membrane. The extraction index of cTnT in three heart failure patients undergoing renal vein catheterization was 8-19%. Kidney function adjusted cTnT levels increased the area under the ROC curve for diagnosis of myocardial infarction of the cTnT analysis in an emergency room cohort. CONCLUSIONS: At high concentrations, often found after a large myocardial infarction, extrarenal clearance of cTnT dominates. At low levels of cTnT, often found in patients with stable cTnT elevations, renal clearance also contribute to the clearance of cTnT. This potentially explains why stable cTnT levels tend to be higher in patients with low kidney function.

Genetic Disruption of Protein Kinase STK25 Ameliorates Metabolic Defects in a Diet-Induced Type 2 Diabetes Model.

Understanding the molecular networks controlling ectopic lipid deposition, glucose tolerance, and insulin sensitivity is essential to identifying new pharmacological approaches to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a negative regulator of glucose and insulin homeostasis based on observations in myoblasts with acute depletion of STK25 and in STK25-overexpressing transgenic mice. Here, we challenged Stk25 knockout mice and wild-type littermates with a high-fat diet and showed that STK25 deficiency suppressed development of hyperglycemia and hyperinsulinemia, improved systemic glucose tolerance, reduced hepatic gluconeogenesis, and increased insulin sensitivity. Stk25(-/-) mice were protected from diet-induced liver steatosis accompanied by decreased protein levels of acetyl-CoA carboxylase, a key regulator of both lipid oxidation and synthesis. Lipid accumulation in Stk25(-/-) skeletal muscle was reduced, and expression of enzymes controlling the muscle oxidative capacity (Cpt1, Acox1, Cs, Cycs, Ucp3) and glucose metabolism (Glut1, Glut4, Hk2) was increased. These data are consistent with our previous study of STK25 knockdown in myoblasts and reciprocal to the metabolic phenotype of Stk25 transgenic mice, reinforcing the validity of the results. The findings suggest that STK25 deficiency protects against the metabolic consequences of chronic exposure to dietary lipids and highlight the potential of STK25 antagonists for the treatment of type 2 diabetes.

Prenatal essential fatty acid deficiency in mice results in long-term gender-specific effects on body weight and glucose metabolism.

Essential fatty acids are important for normal growth and development in early life. However, the long-term effects of prenatal essential fatty acid deficiency (EFAD) on the adult metabolism remain to be determined. The aim of this study was to investigate the effects of an EFAD diet given to mice during late gestation on body weight and body composition, and metabolism in the adult offspring. Pregnant dams were given an EFAD or a control diet during the last 10 days of gestation. After delivery, all mice were fed normal chow and the body weight of the offspring was measured weekly. Furthermore, food intake, energy expenditure and intraperitoneal glucose tolerance were analysed in the adult offspring in addition to body composition (analysed by dual-energy X-ray absorptiometry), plasma levels of leptin, triglycerides and cholesterol. The body weight was lower in the EFAD offspring as compared to the controls during the first 4 weeks of age, and remained lower in the females throughout the study. Lean body mass and plasma leptin levels were also lower in the female EFAD offspring as compared to the controls. Male EFAD offspring were found to have higher fasting glucose and insulin levels as well as higher insulin levels during the glucose tolerance test compared to the controls. However, no differences were found in blood lipids, food intake or energy expenditure between EFAD and control mice of either gender. These results demonstrate that an EFAD diet given during the last 10 days of gestation results in long-term gender-specific effects on body weight and insulin sensitivity in the adult offspring.

Postnatal deficiency of essential fatty acids in mice results in resistance to diet-induced obesity and low plasma insulin during adulthood.

Our objective was to investigate the long-term metabolic effects of postnatal essential fatty acid deficiency (EFAD). Mouse dams were fed an EFAD diet or an isoenergetic control diet 4 days before delivery and throughout lactation. The pups were weaned to standard diet (STD) and were later subdivided into two groups: receiving high fat diet (HFD) or STD. Body composition, energy expenditure, food intake and leptin levels were analyzed in adult offspring. Blood glucose and plasma insulin concentrations were measured before and during a glucose tolerance test. EFAD offspring fed STD were leaner with lower plasma leptin and insulin concentrations compared to controls. EFAD offspring fed HFD were resistant to diet-induced obesity, had higher energy expenditure and lower levels of plasma leptin and insulin compared to controls. These results indicate that the fatty acid composition during lactation is important for body composition and glucose tolerance in the adult offspring.

Blood pressure is the major driving force for plaque formation in aortic-constricted ApoE-/- mice.

OBJECTIVE: Using an aortic constriction model in mice, we studied whether the increase in pressure or the activation of the renin-angiotensin system (RAS) and its main receptors is the main driving force for plaque progression. METHODS: Male ApoE mice underwent sham surgery or placement of a suprarenal silver clip around the aorta (AoC). Half the group was treated with the selective AT1 receptor antagonist losartan (30 mg/kg per day) for 4 weeks. RESULTS: Anesthetized mean arterial pressure (MAP) was increased in AoC mice compared to sham (106 +/- 3 versus 90 +/- 1 mmHg, P < 0.001). Losartan reduced MAP in sham mice (78 +/- 2 mmHg, P < 0.01) but not in AoC (AoC losartan 104 +/- 2 mmHg). Plasma renin concentration (PRC) was increased in AoC mice compared to sham [1.6 +/- 0.3 versus 0.8 +/- 0.2 milliGoldblatt units (mGU)/ml, P < 0.001]. Losartan treatment augmented this difference (18.7 +/- 3.7 versus 4.6 +/- 1.7 mGU/ml, P < 0.01). AT2 receptor mRNA expression was increased 5.8-fold by aortic constriction in thoracic aorta (P < 0.05) and the major site for expression of the AT2 receptor protein was within the plaques. The plaque area was increased in AoC mice compared to sham (0.61 +/- 0.09 versus 0.07 +/- 0.01%, P < 0.001); however, losartan did not alter plaque area. CONCLUSIONS: Our data do not support a role for the AT1 receptor in the progression of atherosclerosis in this model, since blockade with losartan did not alter plaque distribution. Furthermore, we found no support for the counteraction of atherogenesis by increased activity of the RAS acting on the AT2 receptor. Our data suggest that increased pressure is the main driving force for atherosclerosis in this model.

Platelet-derived growth factor B retention is essential for development of normal structure and function of conduit vessels and capillaries.

OBJECTIVE: Extracellular retention of PDGF-B has been proposed to play an important role in PDGF-B signalling. We used the PDGF-B retention motif knockout mouse (RetKO) to study the effects of retention motif deletion on development of micro- and macrovascular structure and function. METHODS: Passive and active properties of conduit vessels were studied using myograph techniques and histological examination. Capillary structure and function was studied using measurements of capillary density in skeletal muscle and by assessing aerobic physical performance in a treadmill setup. Cardiac function was assessed using echocardiography. RESULTS: Myograph experiments revealed an increased diameter and stiffness of the aorta in RetKO. Histological examination showed increased media collagen content and a decreased number of aortic wall layers, however with a similar number of vascular smooth muscle cells. This outward eutrophic remodelling of the aorta was accompanied by endothelial dysfunction. RetKO showed decreased capillary density in skeletal muscle and signs of a defective delivery of capillary oxygen to skeletal muscle, as shown by a decreased physical performance. In RetKO mice, echocardiography revealed an adaptive eccentric cardiac hypertrophy. CONCLUSION: We conclude that retention of PDGF-B during development is essential for a normal conduit vessel function in the adult mouse. Furthermore, PDGF-B retention is also necessary for the development of an adequate capillary density, and thereby for a normal oxygen delivery to skeletal muscle. The lack of primary effects on cardiac function supports the redundant role of PDGF-B in cardiac development.

Endothelial dysfunction in growth hormone transgenic mice.

Acromegaly [overproduction of GH (growth hormone)] is associated with cardiovascular disease. Transgenic mice overexpressing bGH (bovine GH) develop hypertension and hypercholesterolaemia and could be a model for cardiovascular disease in acromegaly. The aims of the present study were to investigate the effects of excess GH on vascular function and to test whether oxidative stress affects endothelial function in bGH transgenic mice. We studied the ACh (acetylcholine)-induced relaxation response in aortic and carotid rings of young (9-11 weeks) and aged (22-24 weeks) female bGH transgenic mice and littermate control mice, without and with the addition of a free radical scavenger {MnTBAP [Mn(III)tetrakis(4-benzoic acid)porphyrin chloride]}. We also measured mRNA levels of eNOS (endothelial nitric oxide synthase) and EC-SOD (extracellular superoxide dismutase). Intracellular superoxide anion production in the vascular wall was estimated using a dihydroethidium probe. Carotid arteries from bGH transgenic mice had an impaired ACh-induced relaxation response (young, 46 +/- 7% compared with 69 +/- 8%; aged, 52 +/- 5% compared with 80 +/- 3%; P < 0.05), whereas endothelial function in aorta was intact in young but impaired in aged bGH transgenic mice. Endothelial dysfunction was corrected by addition of MnTBAP in carotid arteries from young mice and in aortas from aged mice; however, MnTBAP did not correct endothelial dysfunction in carotid arteries from aged bGH transgenic mice. There was no difference in intracellular superoxide anion production between bGH transgenic mice and control mice, whereas mRNA expression of EC-SOD and eNOS was increased in aortas from young bGH transgenic mice compared with control mice (P < 0.05). We interpret these data to suggest that bGH overexpression is associated with a time- and vessel-specific deterioration in endothelial function, initially caused by increased oxidative stress and later by other alterations in vascular function.

Short-term administration of growth hormone (GH) lowers blood pressure by activating eNOS/nitric oxide (NO)-pathway in male hypophysectomized (Hx) rats.

BACKGROUND: The aim of the study was to evaluate the acute and continuous (up to 14 days of treatment) effect of growth hormone (GH) on blood pressure (BP) regulation and to investigate the interplay between GH, nitric oxide (NO) and BP. In un-supplemented and GH supplemented hypophysectomized (Hx) male rats as well as intact rats, continuous resting mean arterial blood pressure (MAP) was measured using telemetry. Baroreceptor activity and the influences of NO on BP control were assessed during telemetric measurement. Furthermore, basal plasma and urine nitrate levels and aortic endothelial nitric oxide synthase (eNOS) expression were analysed. Endothelial function as well as vascular structure in the hindquarter vascular bed was estimated using an in vivo constant-flow preparation. RESULTS: Hypophysectomy was associated with decreased MAP (Hx: 83 +/- 3 vs Intact: 98 +/- 6 mmHg, p < 0.05) and heart rate (HR) (Hx: 291 +/- 4 vs Intact: 351 +/- 7 beat/min, p < 0.05). Endothelial dysfunction and reduced vasculature mass in the hindquarter vascular bed was found in Hx rats. GH substitution caused a further transient decrease in MAP and a transient increase in HR (14% and 16% respectively, p < 0.05). The reduction in MAP appeared to be NO dependent. Aortic eNOS expression was unchanged. GH substitution resulted in an impaired baroreceptor function. Two weeks of GH treatment did not normalise the BP, vascular structure and the endothelial function in the resistance vessels. CONCLUSION: GH substitution seems to have a short lasting effect on lowering blood pressure via activation of the NO-system. An interaction between GH, NO-system and BP regulation can be demonstrated.

Angiotensin II, type 2 receptor is not involved in the angiotensin II-mediated pro-atherogenic process in ApoE-/- mice.

OBJECTIVE: Angiotensin II (Ang II) accelerates atherogenesis in ApoE mice via the angiotensin II, type 1 receptor (AT1) while the type 2 receptor (AT2) is suggested to counteract atherogenesis. To confirm and further explore this possibility, we studied the effect of AT2 receptor antagonism on Ang II-accelerated atherosclerosis. METHODS: ApoE mice were fed a standard or high cholesterol diet (1.25%) for 4 weeks. Mice on each diet were treated with either Ang II (0.5 microg/kg per min) or Ang II in combination with PD123319 (3 mg/kg per day). Plaque distribution was assessed by en face quantification of the thoracic aorta and in cross-sections of the aortic root. Mean arterial pressure (MAP) was measured. AT1 and AT2 receptor expression were analysed using real-time polymerase chain reaction (PCR) and the localization of the AT2 receptor protein confirmed with immunohistochemistry. RESULTS: Ang II infusion increased MAP only in mice on a standard diet (P < 0.001). Regardless of diet, Ang II-infused mice had 22-30 times increased plaque area in the thoracic aorta (P < 0.001 for both). Ang II had no effect on plaque in the aortic root. Plaque area was not affected by PD123319. AT2 receptor was heavily expressed in the plaques and increased six- to ninefold by a high cholesterol diet and Ang II infusion (P < 0.01). CONCLUSION: Ang II increases the extent of atherosclerosis in ApoE mice. Despite up-regulation of the AT2 receptor, we found no support for an effect of the AT2 receptor on atherogenesis in this model.

Haemodynamically significant plaque formation and regional endothelial dysfunction in cholesterol-fed ApoE-/- mice.

Flow-mediated vasodilation is suggested as one of the mechanisms involved in arterial expansive remodelling, which is thought to be a defence mechanism in atherogenesis. In the present study, we tested the hypothesis that lumen obstructive plaque formation is associated with failure of NO (nitric oxide)-dependent vasodilation in conduit vessels. Cardiac function and aortic root flow velocities were assessed using high-resolution echocardiography and two-dimensional-guided pulsed Doppler in ApoE(-/-) (apolipoprotein E-deficient) mice fed a standard or high-cholesterol diet. Endothelial function in the proximal and mid-descending aortic regions was studied using a myograph technique. Flow velocity at the aortic root of cholesterol-fed ApoE(-/-) mice was significantly increased as a result of lumen narrowing, detected via histological analysis. NO-dependent vasodilatory responses were selectively impaired in the atherosclerosis-prone vascular regions in cholesterol-fed ApoE(-/-) mice. In conclusion, consumption of a high-cholesterol diet results in lumen obstructive plaque formation in ApoE(-/-) mice, which significantly alters aortic root haemodynamics. This phenomenon is associated with impaired NO-dependent vasodilation in vessel segments known to be prone to atherosclerosis.

A technique to estimate the rate of whole body nitric oxide formation in conscious mice.

OBJECTIVE: We have established a technique to estimate the total rate of nitric oxide (NO) formation in mice, based on inhalation of a stable oxygen isotope (18O(2)). Changes of NO production with age were also studied. METHODS: The experiments were performed in eight-week- (n=6) and eight-month-old (n=6-7), respectively, female (C57/Bl6xCBAca) mice. Pairs of conscious mice were kept in an air-tight closed system allowing breathing of a mixture containing 18O(2). The 18O(2)-technique was validated by L-NAME (10mg/kg) and lipopolysaccharide (LPS, 8 mg/kg) administration. The concentrations of O(2) and CO(2) in the system were controlled and plasma nitrate analyzed by GC/MS technique. RESULTS: NO formation was similar in young and old mice (young=7.68+/-1.47 vs. old = 6.25+/-1.49 micromol/kg/h, n.s.). Total NO production was reduced after L-NAME treatment in young animals by 91% and in old animals by 71% (p<0.05 for both), whereas LPS administration increased NO production (114+/-17%, p<0.05).Conclusion. NO formation is unaltered with age in mice. The 18O(2)-technique is a valid and specific technique to estimate whole body NO production in conscious mice.

Cardiovascular and renal phenotyping of genetically modified mice: a challenge for traditional physiology.

1. The advent of techniques to genetically modify experimental animals and produce directed mutations in both a conditional and tissue-specific manner has dramatically opened up new fields for physiologists in cardiovascular and renal research. 2. A consequence of altering the genetic background of mice is the difficulty in predicting the phenotypic outcome of the genetic mutation. We therefore suggest that physiologists may need to change their current experimental paradigms to face this new era. Hence, our aim is to propose a complementary research philosophy for physiologists working in the post-genomic era. That is, instead of using strictly hypothesis-driven research philosophies, one will have to perform screening studies of mutant mice, within a field of interest, to find valuable phenotypes. Once a relevant phenotype is found, in-depth studies of the underlying mechanisms should be performed. These follow-up studies should be performed using a traditional hypothesis-driven research philosophy. 3. The rapidly increasing availability of mutated mouse models of human disease also necessitates the development of techniques to characterize these various mouse phenotypes. In particular, the miniaturization and refinement of techniques currently used to study the renal and cardiovascular system in larger animals will be discussed in the present review. Hence, we aim to outline what techniques are currently available and should be present in a laboratory to screen and study renal and cardiovascular phenotypes in genetically modified mice, with particular emphasis on methodologies used in the intact, conscious animal.

GH and IGF-I regulate the expression of endothelial nitric oxide synthase (eNOS) in cardiovascular tissues of hypophysectomized female rats.

OBJECTIVE: This study explored whether short-term replacement therapy with growth hormone (GH) affects blood pressure (BP), heart rate (HR) and endothelial nitric oxide synthase (eNOS) expression in cardiovascular tissues in hypophysectomized (Hx) female rats. DESIGN AND METHODS: BP, HR and the expression of eNOS in the aorta, caval vein and heart were studied in Hx female rats and in Hx female rats that underwent 7 days treatment with GH and thyroxine+glucocorticoids ([T(4)+GC]). Insulin-like growth factor-I (IGF-I) was included in a second experimental protocol to explore the indirect effect of GH. The expression and localisation of eNOS was analysed by immunoblotting and immunohistochemistry. RESULTS: Decreased BP (Hx 98+/-1, Intact 129+/-3 mmHg, P<0.05), HR (Hx 297+/-14, Intact 399+/-31 beats/min, P<0.05) and unchanged eNOS expression was demonstrated in Hx compared with intact rats. None of the hormones affected BP, but both GH and IGF-I increased HR compared with Hx rats (GH 358+/-10, IGF-I 337+/-7, Hx 306+/-11 beats/min, P<0.05). Replacement of GH, GH+[T(4)+GC] and IGF-I resulted in an increased aortic eNOS expression (GH 161+/-24, GH+[T(4)+GC] 177+/-25, IGF-I 153+/-21, Hx 109+/-7%, P<0.05), whereas in caval vein only GH+[T(4)+GC] affected eNOS expression. None of the hormones changed the level of eNOS in the heart. eNOS was localised in the intima layer of the aorta, whereas in the caval vein eNOS was localised in all cell layers. CONCLUSIONS: These findings support the suggested positive role of GH in the regulation of the cardiovascular homeostasis. The observed up-regulation of eNOS, and presumably an increased NO bioavailability, may result in improved endothelial and cardiovascular function.

Brief losartan treatment in young spontaneously hypertensive rats abates long-term blood pressure elevation by effects on renal vascular structure.

OBJECTIVE: We studied the importance of regional vascular structural changes for the long-term antihypertensive effect of brief angiotensin II receptor blockade with losartan in young spontaneously hypertensive rats (SHRs). DESIGN/METHODS: SHRs were treated from 3 to 8 weeks of age with losartan (SHRLos, 30 mg/kg per day in drinking water) or vehicle (SHRCon). Mean arterial blood pressure (MAP) was measured using a telemetric technique from 12 to 25 weeks of age. Indices of vascular structure in the renal and hindquarter limb (HQ) were assessed using a haemodynamic perfusion technique at 13-15 weeks of age. RESULTS: MAP in SHRLos was reduced by 20-30 mmHg throughout the study (P < 0.001) and left ventricular weights were reduced (P < 0.05). The slope of the pressure/flow relationship was significantly changed (P < 0.001) in both kidneys and HQ vascular beds, suggesting greater average lumen dimensions in SHRLos. Pressure-glomerular filtration rate (GFR) curves of SHRLos kidneys were shifted to the left (P < 0.001), suggesting that the reduction in renal vascular resistance was predominantly preglomerular. The changes in structural indices of the heart and HQ closely followed the reduction in MAP. However, resistance at maximal dilatation in SHRLos kidneys was changed out of proportion to the lowering in MAP (P < 0.01). CONCLUSIONS: Brief losartan treatment in young SHRs reduces long-term MAP. The reduced MAP is associated with higher average renal and skeletal muscle vascular dimensions at maximal dilatation, predominantly in the pre-capillary vasculature. The reduction in vascular resistance of the kidney appears to be out of proportion to the reduction in MAP and it may be speculated that this is of primary importance in the long-term hypotensive effect of brief angiotensin II antagonism in SHRs.