Bernard Cosyns

Nanobody-coupled microbubbles as novel molecular tracer

Camelid-derived single-domain antibody-fragments (~15kDa), called nanobodies, are a new class of molecular tracers that are routinely identified with nanomolar affinity for their target and that are easily tailored for molecular imaging and drug delivery applications. We hypothesized that they are well-suited for the design of targeted microbubbles (μBs) and aimed to develop and characterize eGFP- and VCAM-1-targeted μBs. Anti-eGFP (cAbGFP4) and anti-VCAM-1 (cAbVCAM1-5) nanobodies were site-specifically biotinylated in bacteria. This metabolic biotinylation method yielded functional nanobodies with one biotin located at a distant site of the antigen-binding region of the molecule. The biotinylated nanobodies were coupled to biotinylated lipid μBs via streptavidin-biotin bridging. The ability of μB-cAbGFP4 to recognize eGFP was tested as proof-of-principle by fluorescent microscopy and confirmed the specific binding of eGFP to μB-cAbGFP4. Dynamic flow chamber studies demonstrated the ability of μB-cAbVCAM1-5 to bind VCAM-1 in fast flow (up to 5 dynes/cm(2)). In vivo targeting studies were performed in MC38 tumor-bearing mice (n=4). μB-cAbVCAM1-5 or control μB-cAbGFP4 were injected intravenously and imaged using a contrast-specific ultrasound imaging mode. The echo intensity in the tumor was measured 10min post-injection. μB-cAbVCAM1-5 showed an enhanced signal compared to control μBs (p<0.05). Using metabolic and site-specific biotinylation of nanobodies, a method to develop nanobody-coupled μBs was described. The application of VCAM-1-targeted μBs as novel molecular ultrasound contrast agent was demonstrated both in vitro and in vivo.

Role of echocardiography in toxic heart valvulopathy

The notion that drugs can induce valvular heart disease (VHD) has occurred since the 1960s and has received a lot of attention in recent years. This review focuses on different aspects of this distinct valvulopathy in seven sections: (i) historical background, (ii) drug-induced VHD, is this a real entity?, (iii) its morphological and echocardiographic features, (iv) drugs associated with VHD, (v) the influence of cumulative drug dose and risk factors, (vi) the natural course of toxic valvulopathy, and (vii) practical recommendations when using potential valvulopathic drugs.

Echocardiographic integrated backscatter for detecting progression and regression of aortic valve calcifications in rats

BackgroundCalcification is an independent predictor of mortality in calcific aortic valve disease (CAVD). The aim of this study was to evaluate the use of non-invasive, non-ionizing echocardiographic calibrated integrated backscatter (cIB) for monitoring progression and subsequent regression of aortic valvular calcifications in a rat model of reversible renal failure with CAVD, compared to histology.Methods28 male Wistar rats were prospectively followed during 21 weeks. Group 1 (N=14) was fed with a 0.5% adenine diet for 9 weeks to induce renal failure and CAVD. Group 2 (N=14) received a standard diet. At week 9, six animals of each group were killed. The remaining animals of group 1 (N=8) and group 2 (N=8) were kept on a standard diet for an additional 12 weeks. cIB of the aortic valve was calculated at baseline, 9 and 21 weeks, followed by measurement of the calcified area (Ca Area) on histology.ResultsAt week 9, cIB values and Ca Area of the aortic valve were significantly increased in the adenine-fed rats compared to baseline and controls. After 12 weeks of adenine diet cessation, cIB values and Ca Area of group 1 decreased compared to week 9, while there was no longer a significant difference compared to age-matched controls of group 2.ConclusionscIB is a non-invasive tool allowing quantitative monitoring of CAVD progression and regression in a rat model of reversible renal failure, as validated by comparison with histology. This technique might become useful for assessing CAVD during targeted therapy.

IMPACT OF ANESTHESIA ON VALVULAR FUNCTION IN NORMAL RATS DURING ECHOCARDIOGRAPHY

Anesthetic agents have different effects on hemodynamic and cardiac functional parameters. The influence of these changes on valvular function has not been studied in small animals. For this purpose, 48 male Wistar rats were divided into three equal groups. An echocardiogram was performed under inhaled isoflurane 2% gas (group I) or under intraperitoneal pentobarbital 50 mg/kg (group II) or ketamine/xylazine (group III) 40/8 mg/kg. Aortic regurgitation was only found in group III (80%, p < 0.0001 vs. groups I and II). Pulmonary and mitral regurgitation (PR, MR) were observed in all groups but were more frequent in group III (PR 67%, MR 100%) compared with group I (PR 13%, p = 0.003; MR 44%, p = 0.001 vs. group III) and group II (PR 19%, p = 0.011; MR 25%, p < 0.0001 vs. group III). Moreover, valvular regurgitations in group III (except tricuspid regurgitation) were more severe compared with groups I and II. The findings in group III were the result of increased blood pressure and afterload, left ventricular (LV) dilation and decreased function. Also in group III, the regurgitations diminished over time as the blood pressure decreased and LV function recovered. Isoflurane and pentobarbital had less pronounced effects on valvular function (5 and 10 min after induction, respectively) compared with ketamine/xylazine and, therefore, might be the anesthetics of choice for valvular evaluation in male Wistar rats. In conclusion, anesthesia causes hemodynamic changes that may result in functional valvular regurgitations in normal rats.

Evaluation of contractile function and inotropic reserve with tissue velocity, strain and strain rate imaging in streptozotocin-induced diabetes

AIMS The aim of the present study was to evaluate left ventricular (LV) function and contractile reserve (CR) with Doppler myocardial imaging (DMI) in a small animal model for type 1 diabetes. METHODS AND RESULTS Cardiac function was assessed in anaesthetized Wistar rats 6 and 8 weeks after injection of 60 mg/kg of streptozotocin. At 6 weeks of diabetes, colour DMI echocardiography was performed at rest and during incremental dosages of dobutamine (5, 10, 20 microg/kg/min). Left ventricular fractional shortening was decreased after 8 weeks of follow-up [36 +/- 5 (D) vs. 41 +/- 4% (C); P = 0.049]. After 6 weeks of diabetes, DMI measurements were reduced in the diabetic rats in the inferolateral wall at rest [systolic velocity: 2.5 +/- 0.4 (D) vs. 4.4 +/- 0.3 (C) cm/s; P < 0.001; systolic strain rate: 12.2 +/- 3.4 (D) vs. 17.4 +/- 3.2 (C) 1/s; P = 0.012] and during inotropic stimulation [delta velocity (cm/s): 0.2 +/- 0.1 (D) vs. 0.5 +/- 0.3 (C)/5 microg dobutamine; P = 0.002; delta strain rate (1/s): 1.4 +/- 0.9 (D) vs. 3.3 +/- 2.2 (C)/5 microg dobutamine; P = 0.049]. Furthermore, the intraventricular delay in time-to-peak systolic strain was larger in diabetes [20 +/- 18 (D) vs. 10 +/- 7 (C) ms; P= 0.01]. Systolic mitral annular velocity was also lower in the diabetic rats at rest [2.7 +/- 0.4 (D) vs. 3.5 +/- 0.4 (C) cm/s; P < 0.001] and in response to dobutamine [delta velocity (cm/s): 0.1 +/- 0.1 (D) vs. 0.3 +/- 0.2 (C)/5 microg dobutamine; P = 0.013]. CONCLUSION In experimental diabetes, a reduction in radial and longitudinal LV function and CR can be detected with DMI before the onset of a reduced global LV function.

Influence of Heart Rate Reduction on Doppler Myocardial Imaging Parameters in a Small Animal Model

In small animals studies, sick animals often have a significant reduction in heart rate while under anesthesia. The influence of heart rate reduction on Doppler myocardial imaging (DMI) parameters is not known. The aim of the present study was to assess the effect of heart rate reduction on DMI parameters in a small animal model. Twenty-four rats underwent transthoracic echocardiography at baseline and during the administration of ivabradine IV. In all rats, left ventricular (LV) systolic velocity, strain and strain rate were measured in the anteroseptal and inferolateral wall segments from short axis views. In 12 rats (group A), M-mode analysis was also performed for assessment of global LV function. In the other 12 rats (groups B), contractility was quantified invasively using the end-systolic pressure-volume relation (ESPVR) and the preload recruitable stroke work (PRSW). During ivabradine, administration heart rate decreased by 18% in group A (p < 0.001) and 36% in group B (p < 0.001). There was a slight increase in LVEDD and LVESD, with no change in cardiac output or LV ejection fraction. During ivabradine administration, DMI parameters did not change significantly in any group. No significant correlation between DMI parameters and heart rate (r(2) = 0.05) or ejection time (r(2) = 0.14) could be found. The absence of changes in contractility was confirmed by the absence of change in PRSW and end-systolic elastance (Ees). In conclusion, moderate heart rate reduction did not influence DMI measurements in this specific rat model. Therefore, in the interpretation of DMI data when performing small animal studies, moderate heart rate reduction does not need to be taken into account.

Animal models of organic heart valve disease

Heart valve disease is a frequently encountered pathology, related to high morbidity and mortality rates in industrialized and developing countries. Animal models are interesting to investigate the causality, but also underlying mechanisms and potential treatments of human valvular diseases. Recently, animal models of heart valve disease have been developed, which allow to investigate the pathophysiology, and to follow the progression and the potential regression of disease with therapeutics over time. The present review provides an overview of animal models of primary, organic heart valve disease: myxoid age-related, infectious, drug-induced, degenerative calcified, and mechanically induced valvular heart disease.

Occurrence of cardiovascular calcifications in normal, aging rats.

BACKGROUND Cardiovascular calcification is an independent predictor of morbidity and mortality and increases with age. Animal models are frequently used to investigate the underlying pathophysiology. Only scarce data regarding the effect of aging on calcifications in these animal models are available. The aim of this study is to investigate the occurrence of cardiovascular calcifications in normal, aging rats. METHODS A mixed inbred/outbred population of 44 male Lewis/Wistar rats was studied. Group 1 of three-month-old rats, group 2 twelve-month-old, group 3 twenty-four-month-old and group 4 thirty-month-old rats. Calibrated integrated backscatter (cIB) values and blood parameters (creatinine, parathyroid hormone (PTH)) were measured, followed by ex-vivo micro-CT and histology as reference methods. RESULTS Cardiovascular calcifications developed with age, as demonstrated by significantly increasing cIB values of the aortic valve and myocardium. This was confirmed by a significant increase in the calcified volume on ex-vivo micro-CT and in the histological calcium score. There was also a significantly higher level of creatinine and PTH with age. CONCLUSIONS As in humans, cardiovascular calcifications progressively increase with age in the normal rat. Therefore the aging rat model could be used for studying calcifying cardiovascular disease. cIB might have a value in future studies for the early detection of subclinical calcifications in humans.

Echocardiographic and histological assessment of age-related valvular changes in normal rats.

Aging is associated with morphologic and functional alterations of the rats left ventricle. However, the time-course of valvular function and morphology in normal aging rats has not yet been studied. For this purpose, 30 male Wistar rats (318 +/- 5g, 10 weeks old) underwent serial echocardiograms for 58 weeks under sodium pentobarbital 50 mg/kg IP anesthetization followed by necropsy. Histopathology was also performed in two additional groups of 10 rats at 10 and 30 weeks of age. Regurgitations were considered as any retrograde flow on 2-D or M-mode color Doppler echocardiography. Tricuspid regurgitation was already found at 10 weeks of age and became more frequent with age. Pulmonary, mitral and aortic regurgitation was seldom observed at 10 weeks but became more frequent after 30 weeks. For the mitral and aortic valve, this was also associated with an increase in valvular thickness because of nodular or segmental myxoid leaflet changes. The severity of valvular regurgitations did not increase with age. In conclusion, aging leads to morphologic and functional valvular changes in normal rats. This is important when investigating models of valvular heart disease in small animals.

Contrast harmonic imaging improves the evaluation of left ventricular function in ventilated patients: comparison with transesophageal echocardiography

AIMS The study examined the value of contrast echocardiography (CE) in the assessment of left ventricular (LV) wall motion in ventilated patients in comparison with transesophageal (TOE) and standard fundamental transthoracic imaging (SE). METHODS Transthoracic echocardiograms were done in 40 ventilated patients. Wall motion was evaluated using the recommendations of the American Society of Echocardiography on SE, CE and TOE. A visualization score was assigned on a scale of 2-0 for each of 16 segments. The segment was assigned a value of 2 if the segment was seen in both systole and diastole, 1 if seen only in systole or diastole, and 0 if not seen at all. A confidence score was also given for each segment with each technique (unable to evaluate; not sure; sure). The ejection fraction (EF) was estimated visually for each technique, and a confidence score was also applied to the EF. RESULTS Visualization score 0 was present in 6.2 segments/patient on SE, 1.2 on CE (P<0.0001) and 1.1 on TOE (P<0.0001). An average of 6.5 segments were read with surety on SE, 11.5 on CE (P<0.0001) and 12.3 on TOE ( P<0.0001 ). There was no significant difference for CE vs TOE. EF was uninterpretable in 32% on SE, 0% on CE (P<0.001 and 0% on TOE (P<0.001). The EF was read with surety in 53% of patients on SE, 88% on CE (P < 0.0001) and 93% with TOE (P<0.0001) with no difference for CE vs TOE. Thus, wall motion was seen with more confidence on CE and TOE. CONCLUSIONS In the ventilated patients with suboptimal transthoracic echocardiograms for the evaluation of the LV function, CE provides image quality of regional and global LV function similar to that achieved with TOE echocardiography.