Non-invasive ultrasound-based cardiovascular imaging in mouse models of atherosclerosis

Atherosclerosis is a chronic multi-factorial vascular disease. It generally requires large clinical settings and over many years to study the disease progression in man. Genetically modified mouse models of atherosclerosis have dramatically increased research feasibility within this area. However, for optimal translational studies, it is increasingly important to explore how human-like these atherosclerotic mouse models demonstrate their disease phenotype and respond to established cardiovascular interventions. The aims of this thesis were to develop and apply in vivo translational techniques to study living atherosclerotic mice, with human-relevant disease phenotype. Color Doppler guided echocardiography and a high-frequency ultrasound biomicroscope were used for imaging of peripheral and coronary artery function and morphology. The potential role of IL-18 in mice was explored in relationship to mouse coronary artery disease. Finally temporal effects of rosuvastatin on cardiovascular phenotype were studied in an ApoE knockout mouse model of atherosclerosis. This thesis illustrates that it is possible to investigate both central and peripheral atherosclerosis in living mice using ultrasound-based techniques. Our findings may also suggest an important role of IL-18 in late stage atherosclerosis in an advanced model of atherosclerosis. Finally, this particular ApoE knockout mouse model showed time-dependent beneficial cardiovascular effects following rosuvastatin treatment. The established translational functional and morphological imaging platform in combination with our human-like statin-responding mouse model, provide us with powerful tools for future atherosclerosis research

Increasing peripheral artery intima thickness from childhood to seniority

Background - Using new, very high-resolution ultrasound biomicroscopy, we examined the thickness of artificial layers of silicone and intima thickness (IT) of radial and anterior tibial arteries in healthy subjects and in patients with vascular disease. Methods and Results - Silicone layers of varying thicknesses and mesenteric artery specimens obtained from 18 patients undergoing colectomy were measured by both ultrasound biomicroscopy (55 MHz) and morphometry. There was high correlation (r > 0.9; P < 0.0001) between IT and intima area versus ultrasound biomicroscopy. In 90 healthy subjects (aged between 10 and 90 years), radial and anterior tibial arterial IT and intima-media thickness were measured, as was carotid intima-media thickness in 56 of these subjects. Age was strongly related with both media thickness and IT of both peripheral arteries. Correlations were found between carotid intima-media thickness and both radial and anterior tibial IT/intima-media thickness (r=0.44 to 0.53; P < 0.0001). The IT-to-lumen diameter ratio increased with age and was larger at all ages in the anterior tibial artery (0.067 +/- 0.034) versus the radial artery (0.036 +/- 0.012; P < 0.0001). A thicker radial intimal layer was found in patients with peripheral artery disease. Conclusion - This study is the first to our knowledge in humans to show the feasibility of measuring IT of the radial and anterior tibial arteries using very high-resolution ultrasound. IT progresses with age, and the IT-to-lumen diameter ratio is largest in the arteries of the foot. Assessment of IT by ultrasound biomicroscopy may aid in detecting early peripheral vascular abnormalities

Deficiency of filamin A in endothelial cells impairs left ventricular remodelling after myocardial infarction

Aims: Actin-binding protein filamin A (FLNA) regulates signal transduction important for cell locomotion, but the role of FLNA after myocardial infarction (MI) has not been explored. The main purpose of this study was to determine the impact of endothelial deletion of FLNA on post-MI remodelling of the left ventricle (LV). Methods and results: We found that FLNA is expressed in human and mouse endothelial cells (ECs) during MI. To determine the biological significance of endothelial expression of FLNA, we used mice that are deficient for endothelial FLNA by cross-breeding adult mice expressing floxed Flna (Flnao/fl) with mice expressing Cre under the vascular endothelial-specific cadherin promoter (VECadCre+). Male Flnao/fl and Flnao/fl/VECadCre+ mice were subjected to permanent coronary artery ligation to induce MI. Flnao/fl/VECadCre+ mice that were deficient for endothelial FLNA exhibited larger and thinner LV with impaired cardiac function as well as elevated plasma levels of NT-proBNP and decreased secretion of VEGF-A. The number of capillary structures within the infarcted areas was reduced in Flnao/fl/VECadCre+ hearts. ECs silenced for Flna mRNA expression exhibited impaired tubular formation and migration, secreted less VEGF-A, and produced lower levels of phosphorylated AKT and ERK1/2 as well as active RAC1. Conclusion: Deletion of FLNA in ECs aggravated MI-induced LV dysfunction and cardiac failure as a result of defective endothelial response and increased scar formation by impaired endothelial function and signalling

SGLT2 inhibition with empagliflozin improves coronary microvascular function and cardiac contractility in prediabetic ob/ob−/− mice

Abstract Background Sodium-glucose cotransporter 2 inhibitors (SGLT2i) is the first class of anti-diabetes treatment that reduces mortality and risk for hospitalization due to heart failure. In clinical studies it has been shown that SGLT2i’s promote a general shift to fasting state metabolism characterized by reduced body weight and blood glucose, increase in glucagon/insulin ratio and modest increase in blood ketone levels. Therefore, we investigated the connection between metabolic changes and cardiovascular function in the ob/ob−/− mice; a rodent model of early diabetes with specific focus on coronary microvascular function. Due to leptin deficiency these mice develop metabolic syndrome/diabetes and hepatic steatosis. They also develop cardiac contractile and microvascular dysfunction and are thus a promising model for translational studies of cardiometabolic diseases. We investigated whether this mouse model responded in a human-like manner to empagliflozin treatment in terms of metabolic parameters and tested the hypothesis that it could exert direct effects on coronary microvascular function and contractile performance. Methods Lean, ob/ob−/− untreated and ob/ob−/− treated with SGLT2i were followed for 10 weeks. Coronary flow velocity reserve (CFVR) and fractional area change (FAC) were monitored with non-invasive Doppler ultrasound imaging. Food intake, urinary glucose excursion and glucose control via HbA1c measurements were followed throughout the study. Liver steatosis was assessed by histology and metabolic parameters determined at the end of the study. Results Sodium-glucose cotransporter 2 inhibitors treatment of ob/ob−/− animals resulted in a switch to a more catabolic state as observed in clinical studies: blood cholesterol and HbA1c were decreased whereas glucagon/insulin ratio and ketone levels were increased. SGLT2i treatment reduced liver triglyceride, steatosis and alanine aminotransferase, an indicator for liver dysfunction. l-Arginine/ADMA ratio, a marker for endothelial function was increased. SGLT2i treatment improved both cardiac contractile function and coronary microvascular function as indicated by improvement of FAC and CFVR, respectively. Conclusions Sodium-glucose cotransporter 2 inhibitors treatment of ob/ob−/− mice mimics major clinical findings regarding metabolism and cardiovascular improvements and is thus a useful translational model. We demonstrate that SGLT2 inhibition improves coronary microvascular function and contractile performance, two measures with strong predictive values in humans for CV outcome, alongside with the known metabolic changes in a preclinical model for prediabetes and heart failure

Impaired Coronary and Renal Vascular Function in Spontaneously Type 2 Diabetic Leptin-Deficient Mice

<div><p>Background</p><p>Type 2 diabetes is associated with macro- and microvascular complications in man. Microvascular dysfunction affects both cardiac and renal function and is now recognized as a main driver of cardiovascular mortality and morbidity. However, progression of microvascular dysfunction in experimental models is often obscured by macrovascular pathology and consequently demanding to study. The obese type 2 diabetic leptin-deficient (ob/ob) mouse lacks macrovascular complications, i.e. occlusive atherosclerotic disease, and may therefore be a potential model for microvascular dysfunction. The present study aimed to test the hypothesis that these mice with an insulin resistant phenotype might display microvascular dysfunction in both coronary and renal vascular beds.</p><p>Methods and Results</p><p>In this study we used non-invasive Doppler ultrasound imaging to characterize microvascular dysfunction during the progression of diabetes in ob/ob mice. Impaired coronary flow velocity reserve was observed in the ob/ob mice at 16 and 21 weeks of age compared to lean controls. In addition, renal resistivity index as well as pulsatility index was higher in the ob/ob mice at 21 weeks compared to lean controls. Moreover, plasma L-arginine was lower in ob/ob mice, while asymmetric dimethylarginine was unaltered. Furthermore, a decrease in renal vascular density was observed in the ob/ob mice.</p><p>Conclusion</p><p>In parallel to previously described metabolic disturbances, the leptin-deficient ob/ob mice also display cardiac and renal microvascular dysfunction. This model may therefore be suitable for translational, mechanistic and interventional studies to improve the understanding of microvascular complications in type 2 diabetes.</p></div

Representative color Doppler image for measurement of coronary flow Velocities.

<p>Typical recordings of resting and hyperaemic flow velocity measurement in the left coronary artery were performed with color Doppler ultrasound in lean and leptin-deficient (ob/ob) mice at 10, 16 and 21weeks of age. Hypereamic flow velocity was induced by intravenous infusion of adenosine (140 μg/kg/min). Coronary flow velocity reserve was calculated as the ratio of peak diastolic flow velocities (red line) before (resting) and during (hyperaemic) adenosine infusion. A: Resting coronay flow velocity in lean mice. B: Hyperaemic coronary flow velocity in lean mice. C: Resting coronary flow velocity in ob/ob mice. D: Hyperaemic coronary flow velocity in ob/ob mice.</p