Colin C. Schwarzwald

AntagomiR directed against miR-20a restores functional BMPR2 signalling and prevents vascular remodelling in hypoxia-induced pulmonary hypertension

AIMS Dysregulation of the bone morphogenetic protein receptor type 2 (BMPR2) is a hallmark feature that has been described in several forms of pulmonary hypertension. We recently identified the microRNA miR-20a within a highly conserved pathway as a regulator of the expression of BMPR2. To address the pathophysiological relevance of this pathway in vivo, we employed antagomiR-20a and investigated whether specific inhibition of miR-20a could restore functional levels of BMPR2 and, in turn, might prevent pulmonary arterial vascular remodelling. METHODS AND RESULTS For specific inhibition of miR-20a, cholesterol-modified RNA oligonucleotides (antagomiR-20a) were synthesized. The experiments in mice were performed by using the hypoxia-induced mouse model for pulmonary hypertension and animal tissues were analysed for right ventricular hypertrophy and pulmonary arterial vascular remodelling. Treatment with antagomiR-20a enhanced the expression levels of BMPR2 in lung tissues; moreover, antagomiR-20a significantly reduced wall thickness and luminal occlusion of small pulmonary arteries and reduced right ventricular hypertrophy. To assess BMPR2 signalling and proliferation, we performed in vitro experiments with human pulmonary arterial smooth muscle cells (HPASMCs). Transfection of HPASMCs with antagomiR-20a resulted in activation of downstream targets of BMPR2 showing increased activation of Id-1 and Id-2. Proliferation of HPASMCs was found to be reduced upon transfection with antagomiR-20a. CONCLUSION This is the first report showing that miR-20a can be specifically targeted in an in vivo model for pulmonary hypertension. Our data emphasize that treatment with antagomiR-20a restores functional levels of BMPR2 in pulmonary arteries and prevents the development of vascular remodelling.

Comparison of unfractioned and low molecular weight heparin for prophylaxis of coagulopathies in 52 horses with colic: a randomised double-blind clinical trial.

REASONS FOR PERFORMING STUDY Unfractioned heparin (UFH) is widely used for prophylaxis of coagulation disorders, especially in colic-affected horses. However, it is accompanied by certain side effects. OBJECTIVES To compare the efficacy and side effects of unfractioned and low molecular weight heparin (LMWH) in horses with colic. METHODS The study was carried out as a randomised, double-blind, controlled clinical trial. Fifty-two horses with colic were treated subcutaneously with either UFH (heparin calcium, 150 iu/kg bwt initially, followed by 125 iu/kg bwt q. 12 h for 3 days and then 100 iu/kg bwt q. 12 h) or LMWH (dalteparin, 50 iu/kg bwt q. 24 h). All horses underwent daily physical examination including assessment of jugular veins, local reaction to heparin injections, haematological evaluation and coagulation profiles over up to 9 days. RESULTS The type of heparin used did not affect the general behaviour and condition. There were significantly more jugular vein changes in horses treated with UFH. Packed cell volume decreased significantly within the first few days of UFH treatment, but did not change significantly in horses treated with LMWH. Activated partial thromboplastin time (aPTT) and thrombin time (TT) were prolonged in horses treated with UFH but not in those treated with LMWH. CONCLUSIONS It was concluded that, in comparison to UFH, LMWH has markedly fewer side effects in horses. POTENTIAL RELEVANCE Therefore, LMWH is recommended for prophylaxis of coagulation disorders in colic patients.

Recommendations for management of equine athletes with cardiovascular abnormalities.

Murmurs and arrhythmias are commonly detected in equine athletes. Assessing the relevance of these cardiovascular abnormalities in the performance horse can be challenging. Determining the impact of a cardiovascular disorder on performance, life expectancy, horse and rider or driver safety relative to the owners future expectations is paramount. A comprehensive assessment of the cardiovascular abnormality detected is essential to determine its severity and achieve these aims. This consensus statement presents a general approach to the assessment of cardiovascular abnormalities, followed by a discussion of the common murmurs and arrhythmias. The description, diagnosis, evaluation, and prognosis are considered for each cardiovascular abnormality. The recommendations presented herein are based on available literature and a consensus of the panelists. While the majority of horses with cardiovascular abnormalities have a useful performance life, periodic reexaminations are indicated for those with clinically relevant cardiovascular disease. Horses with pulmonary hypertension, CHF, or complex ventricular arrhythmias should not be ridden or driven.

Quantitative analysis of stress echocardiograms in healthy horses with 2-dimensional (2D) echocardiography, anatomical M-mode, tissue Doppler imaging, and 2D speckle tracking.

BACKGROUND Stress echocardiography is used to diagnose myocardial dysfunction in horses, but current methods are not well standardized. The influence of heart rate (HR) on measurements is largely unknown. OBJECTIVES To investigate the use of 2-dimensional echocardiography (2DE), anatomical M-mode (AMM), tissue Doppler imaging (TDI), and 2D speckle tracking (2DST) at rest and after exercise for quantification of regional and global left-ventricular (LV) function. ANIMALS Five athletic Warmblood horses; 11.6+/-3.6 years; 529+/-48 kg. METHODS Prospective study. Three separate echocardiographic examinations were performed before (baseline) and over 5 minutes after treadmill exercise with 2DE (1st, short-axis view; 2nd, long-axis view) and pulsed-wave TDI (3rd examination). Offline analyses were performed at baseline and after exercise at HR 120, 110, 100, 90, and 80 minute(-1). Global and segmental measurements were compared by analysis of variance. RESULTS Quantitative analyses of stress echocardiograms were feasible in all horses. None of the AMM indices changed significantly after exercise. Stroke volume and ejection fraction by 2DE and strain by 2DST decreased, whereas strain rate by 2DST increased significantly at HR>100 minute(-1). TDI analyses were technically difficult and provided little additional information. CONCLUSIONS AND CLINICAL IMPORTANCE Volumetric indices by 2DE and strain and strain rate by 2DST are applicable for quantitative assessment of stress echocardiograms. In healthy horses, they are significantly altered at a HR>100 minute(-1) and need to be evaluated in view of the instantaneous HR. Further investigations are needed to define the clinical value of stress echocardiography in horses with cardiac disease.

Left Ventricular Radial and Circumferential Wall Motion Analysis in Horses Using Strain, Strain Rate, and Displacement by 2D Speckle Tracking

BACKGROUND Noninvasive assessment of left-ventricular (LV) function is clinically relevant, but is incompletely studied in horses. OBJECTIVES To document the feasibility, describe the techniques, and determine the reliability of 2D speckle tracking (2DST) for characterization of LV radial and circumferential wall motion in horses. ANIMALS Three Standardbreds, 3 Thoroughbreds; age 8-14 years; body weight 517-606 kg. METHODS Observational study. Repeated 2-dimensional echocardiographic examinations were performed in unsedated horses by 2 observers and subsequently analyzed by 2DST. Test reliability was determined for segmental and for averaged 2DST indices (including strain, strain rate, displacement, and rotation) by estimating measurement variability, within-day interobserver variability, between-day interobserver variability, and between-day intraobserver variability. Variability was expressed as coefficient of variation (percent) and the absolute value below which the difference between 2 measurements will lie with 95% probability. RESULTS 2DST analyses were feasible in 16 of 18 echocardiographic studies. The automated tracking was accurate during systole but inaccurate during diastole. Reliability was higher for radial compared to circumferential measurements. For radial strain, radial systolic strain rate, and radial systolic displacement, the test-retest variabilities ranged between 2.4 and 33.1% for segmental and between 4.1 and 16.1% for averaged measurements. CONCLUSIONS AND CLINICAL IMPORTANCE Systolic radial motion of the LV at the chordal level could be reliably characterized in horses by 2DST. Circumferential measurements were less reliable. Diastolic measurements were invalid because of inaccurate tracking. The clinical value of LV wall motion analysis by 2DST in horses requires further investigation.

Methods and Reliability of Tissue Doppler Imaging for Assessment of Left Ventricular Radial Wall Motion in Horses

BACKGROUND Noninvasive assessment of left ventricular (LV) function is incompletely studied in horses. OBJECTIVES The goals of this study were to investigate the feasibility, techniques, and reliability of tissue Doppler imaging (TDI) for characterization of LV radial wall motion in healthy horses. ANIMALS Three Standardbreds, 3 Thoroughbreds; age 8-14 years; body weight 517-606 kg. METHODS Repeated echocardiographic examinations were performed by 2 observers in unsedated horses using TDI. Test reliability was determined by estimating measurement variability, within-day interobserver variability, and between-day interobserver and intraobserver variability of all echocardiographic variables. Variability was expressed as coefficient of variation (CV) and the absolute value below which the difference between 2 measurements will lie with 95% probability. RESULTS Assessment of LV radial wall motion by TDI was feasible in all horses. Measurement variabilities were very low (CV < 5%) to low (CV 5-15%) for most variables. Within-day interobserver variability as well as between-day interobserver and intraobserver variabilities were low to moderate (CV 16-25%) for most variables. All pulsed-wave TDI variables of systolic LV function showed very low to low variability, whereas some of the variables of LV diastolic and LA function showed moderate to high (CV > 25%) variability. Pulsed-wave TDI variables appeared more reliable than color TDI variables. CONCLUSIONS AND CLINICAL IMPORTANCE Measurement of TDI indices of LV function is feasible and reliable in adult Standardbred and Thoroughbred horses. The clinical relevance of LV function assessment by TDI remains to be determined.

EcPV2 DNA in equine papillomas and in situ and invasive squamous cell carcinomas supports papillomavirus etiology

Equine penile papillomas, in situ carcinomas, and invasive carcinomas are hypothesized to belong to a continuum of papillomavirus-induced diseases. The former ones clinically present as small grey papules, while the latter 2 lesions are more hyperplasic or alternatively ulcerated. To test the hypothesis that these lesions are papillomavirus-induced, samples of 24 horses with characteristic clinical and histologic findings of penile papillomas or in situ or invasive squamous cell carcinomas were collected. As controls, 11 horses with various lesions—namely, Balanoposthitis (6 cases), melanoma (3 cases), follicular cyst (1 case), and amyloidosis (1 case)—were included. DNA was extracted and polymerase chain reaction applied to amplify papillomavirus DNA. The respective primers were designed to amplify DNA of the recently discovered equine papillomavirus EcPV2. All tested papilloma and squamous cell carcinoma samples were found to contain DNA of either of 2 previously published EcPV2 variants. Among the other samples 6 of 11 were found to contain EcPV2 DNA. To further support the findings and to determine where the papillomavirus DNA was located within the lesions, an in situ hybridization for the detection of EcPV2 DNA was established. The samples tested by this technique were found to clearly contain papillomavirus nucleic acid concentrated in the nucleus of the koilocytes. The findings of this study support previous data and the hypothesis that papillomaviruses induce the described penile lesions in horses.

Plasma atrial natriuretic peptide concentrations in horses with heart disease: a pilot study.

Atrial natriuretic peptide (ANP) is a cardiovascular biomarker that might be useful in assessing the severity of cardiac disease in horses. Plasma ANP concentrations (Cp(ANP)) were compared between horses with heart disease but normal chamber size and function (Group A; n=6), horses with heart disease associated with left atrial (LA) enlargement, LA dysfunction, and/or left ventricular (LV) enlargement (Group B; n=5), and horses with no clinically apparent cardiovascular disease (Group C; n=13). The median (min-max) for Cp(ANP) was significantly higher in Group B (53.5 (36.0-70.7) pg/mL), compared to Group A (12.5 (6.3-19.8) pg/mL) and Group C (13.4 (7.2-34.0) pg/mL). Backwards stepwise multiple linear regression showed that Cp(ANP) in horses with heart disease was related to LA dimensions, but not to LV size, LA function, and LV function. The results indicated that Cp(ANP) in horses might be useful in detecting LA enlargement and that Cp(ANP) could be related to the severity of cardiac disease. Larger prospective studies are necessary to confirm these results.

Effects on cardiopulmonary function and oxygen delivery of doses of romifidine and xylazine followed by constant rate infusions in standing horses.

The objective of this study was to compare the cardiopulmonary effects of a xylazine or romifidine loading-dose, followed by a constant rate infusion (CRI) of the same α(2)-agonist. Nine research horses were treated in a randomized, blinded, crossover design with xylazine or romifidine. After instrumentation, a loading dose of intravenous xylazine (1mg/kg) or romifidine (80μg/kg) was administered, immediately followed by a CRI of xylazine (0.69mg/kg/h) or romifidine (30μg/kg/h) for a duration of 2h. Cardiopulmonary variables were recorded before bolus administration, during CRI, and for 1h after discontinuing drug administration. A significant decrease in haemoglobin concentration (tHb), arterial oxygen content (CaO(2)), oxygen delivery (D˙O(2)), mixed venous partial pressure of oxygen, heart rate, and cardiac output (Q˙t) followed the loading dose with both treatments. Carotid arterial blood pressure (ABP), systemic vascular resistance, and right atrial pressure (RAP) increased significantly. The increased ABP was followed by a significant decrease compared to baseline. Mean pulmonary arterial pressure increased significantly with romifidine only. No significant changes in stroke volume, arterial partial pressure of oxygen, and oxygen consumption were observed. Changes in Q˙t and RAP were more pronounced with romifidine. During CRI, tHb, and CaO(2) were significantly higher with romifidine, whereas D˙O(2) did not differ between treatments. Overall, cardiopulmonary effects were more pronounced and lasted longer with romifidine compared to xylazine. However, during CRI, there was no difference in D˙O(2) between drugs. With both α(2)-agonists, cardiovascular effects were most pronounced after loading dose administration and tended to stabilize during CRI.

Sequential L-lactate concentration in hospitalised equine neonates: A prospective multicentre study

REASONS FOR PERFORMING STUDY Evaluation of serial blood lactate concentrations [LAC] are of prognostic value for morbidity and mortality in critically ill human patients and neonatal foals, but have not been prospectively evaluated in a large multicentre study of critically ill neonatal foals. OBJECTIVES To prospectively evaluate the prognostic value of sequential [LAC] analysis in critically ill neonatal foals with risk of mortality. STUDY DESIGN Prospective, observational study. METHODS Thirteen university and private equine referral hospitals enrolled 643 foals over the 2008 foaling season and [LAC] was measured at admission ([LAC]ADMIT ) and 24 ([LAC]24 ), 48 ([LAC]48 ), 72 ([LAC]72 ), 96 ([LAC]96 ) and 120 h ([LAC]120 ) after admission. [LAC] changes over time ([LAC]Δ) were calculated between sampling points. RESULTS Nonsurvivors had significantly greater [LAC]ADMIT , [LAC]24 and [LAC]48 compared with surviving foals (P<0.001). In nonsurviving foals [LAC]Δ did not decrease over time while survivors showed significant positive [LAC]Δ between [LAC]ADM -24 and all other time periods (P<0.001). Logistic regression analysis showed that the odds of survival decreased for each 1 mmol/l [LAC] increase at all time points for all critically ill foals, independent of major final diagnoses as potential confounders. Septic foals had significantly greater [LAC] at all time points compared with nonseptic foals (P<0.001) and [LAC]Δ in septic foals was significantly more positive (suggesting better clearance of lactate from the blood) only at [LAC]ADM -24 and [LAC]72-96 (P<0.01), while in nonseptic foals [LAC]Δ was significantly positive between [LAC]ADM -24 compared with all other time periods (P<0.001). CONCLUSIONS Blood lactate concentration is a strong, independent biomarker used to predict mortality in critically ill foals. Lactate metabolism is impaired in nonsurviving and septic foals and [LAC]Δ can be utilised to identify patients at high risk for mortality.