Helen Poser

Decellularized Allogeneic Heart Valves Demonstrate Self-Regeneration Potential after a Long-Term Preclinical Evaluation

Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT) reconstruction in Vietnamese Pigs (n = 11) with 6 (n = 5) and 15 (n = 6) follow-up months. Repositioned native valves (n = 2 for each time) were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly higher number and specific topographic distribution in respect to control valves. Following the longest follow-up ever realized in preclinical models, non-preconditioned decellularized allogeneic valves offer suitable microenvironment for in vivo cell homing and tissue remodeling. Manufactured with simple, timesaving and cost-effective procedures, these promising valve replacements hold promise to become an effective alternative, especially for pediatric patients.

Physiological Performance of a Detergent Decellularized Heart Valve Implanted for 15 Months in Vietnamese Pigs: Surgical Procedure, Follow‐up, and Explant Inspection

This study features the longest experimental follow-up for decellularized heart valves implanted in an animal model. Porcine aortic heart valves were decellularized according to a disclosed standardized method in which TRITON X-100 and sodium cholate (TRICOL) are used in succession, followed by a further treatment with the endonuclease Benzonase to completely remove the nucleic acid remnants. Experimental animals (n = 17), represented by Vietnamese pigs (VPs), received a decellularized aortic allograft as a substitute for the replacement of their right ventricular outflow tract. The surgical implantation of the TRICOL-treated aortic valve conduit was successful in 11 VPs, while perioperative or postoperative complications occurred in the remaining six animals. In the sham-operated group (n = 4), the native pulmonary root was excised and immediately reimplanted orthotopically in the same animal. Echocardiography demonstrated a satisfactory hemodynamic performance of the TRICOL-treated valves during follow-up as well as the absence of relevant leaflet alterations concerning thickness and motility or valve insufficiency. At explantation, macroscopic inspection of tissue-engineered heart valve conduits did not evidence calcifications and showed a decreased wall thickness, comparable to that of the reimplanted native pulmonary roots. Noteworthy, extended functional performance, recovery of DNA content, and active extracellular matrix precursor incorporation are apparently compatible with the properties of a living self-supporting substitute.

Red blood cell distribution width in dogs with chronic degenerative valvular disease.

OBJECTIVE To evaluate RBC distribution width (RDW) in dogs with chronic degenerative valvular disease (CDVD) with compensated or decompensated heart failure. DESIGN Retrospective case-control study. ANIMALS 27 healthy dogs and 135 dogs with CDVD (87 dogs with compensated heart failure and 48 dogs with decompensated heart failure). PROCEDURES The RDW and various CBC and serum biochemical variables were compared among groups. Correlations between RDW and various echocardiographic variables were evaluated. RESULTS Mean ± SD RDW in dogs with CDVD (13.1% ± 1.0%) was not significantly different from that of healthy dogs (12.8% ± 0.8%). The RDW of dogs with CDVD and compensated heart failure (13.0% ± 1.0%) was not significantly different from that of dogs with CDVD and decompensated heart failure (13.2% ± 1.1%). The RDW had a significant, weak, negative correlation with Hct (correlation coefficient, -0.250), hemoglobin concentration (correlation coefficient, -0.219), and mean corpuscular volume (correlation coefficient, -0.211). The RDW had a significant, weak, positive correlation with 1 echocardiographic index of CDVD severity (ie, the left atrium-to-aorta ratio [correlation coefficient, 0.183]). CONCLUSIONS AND CLINICAL RELEVANCE In this study population, RDW did not seem to be associated with the presence of heart failure or CDVD.

Tricuspid annular plane systolic excursion in dogs with myxomatous mitral valve disease with and without pulmonary hypertension

OBJECTIVES To evaluate tricuspid annular plane systolic excursion (TAPSE) in dogs with myxomatous mitral valve disease (MMVD) with or without pulmonary hypertension (PH) and to study the correlations with clinical and echocardiographic parameters. ANIMALS The study population included 99 dogs with MMVD and tricuspid regurgitation. METHODS This is a prospective clinical study. All dogs received a transthoracic echocardiographic evaluation, including 2D, M-mode, echo-Doppler, and tissue Doppler measurements. The TAPSE was measured from the left apical four-chamber view and normalized for the effect of body weight (nTAPSE). The dogs were grouped according to the severity of MMVD (American College of Veterinary Internal Medicine guidelines) and presence/absence and severity of PH. Significant differences between TAPSE or nTAPSE and echocardiographic parameters were analyzed among the MMVD and PH severity groups. Correlations between TAPSE or nTAPSE and echocardiographic parameters were calculated. RESULTS Tricuspid annular plane systolic excursion or nTAPSE were not significantly different among dogs of the MMVD or PH severity groups. Significant correlations were obtained between TAPSE and body weight, left ventricular and atrial dimensions, early diastolic septal and early diastolic and systolic tricuspid annulus velocity (p<0.001); nTAPSE was significantly correlated with normalized end-diastolic left ventricular dimension and fractional shortening (p<0.001). CONCLUSIONS The results show that neither TAPSE nor nTAPSE are reduced in dogs with MMVD with or without PH. It remains unclear if the right ventricle function is not reduced or if a reduced right ventricle function is masked by the contraction of the left ventricle through ventricular interdependence.

Two-dimensional, M-mode and Doppler-derived echocardiographic parameters in sedated healthy growing female sheep:

Despite the fact that sheep are a widely used animal model in cardiovascular research, reference values for transthoracic echocardiography in normal growing animals are not available. Eight healthy female lambs underwent two-dimensional, M-mode and pulsed wave Doppler echocardiographic examination at 100 days of age and every three months thereafter over a 12-month period. The study was conducted under sedation with midazolam, butorphanol and constant rate infusion of intravenous propofol. Their growth phase was completed at about one year of age. All the echocardiographic parameters considered were significantly correlated with body weight and age class except for the left ventricular systolic and diastolic diameters. Functional indices were not correlated to body weight or age except for the E-point to septal separation distance (EPSS). Doppler-derived parameters were not influenced by independent variables. Transthoracic echocardiography can be considered an applicable method for cardiovascular research using a growing lamb animal model after appropriate adjustments for age and body size.

Utility of Tissue Doppler Imaging in the Echocardiographic Evaluation of Left and Right Ventricular Function in Dogs with Myxomatous Mitral Valve Disease with or without Pulmonary Hypertension

Background In human medicine, right ventricular (RV) functional parameters represent a tool for risk stratification in patients with congestive heart failure caused by left heart disease. Little is known about RV alterations in dogs with left‐sided cardiac disorders. Objectives To assess RV and left ventricular (LV) function in dogs with myxomatous mitral valve disease (MMVD) with or without pulmonary hypertension (PH). Animals One‐hundred and fourteen dogs: 28 healthy controls and 86 dogs with MMVD at different stages. Methods Prospective observational study. Animals were classified as healthy or having MMVD at different stages of severity and according to presence or absence of PH. Twenty‐eight morphological, echo‐Doppler, and tissue Doppler imaging (TDI) variables were measured and comparison among groups and correlations between LV and RV parameters were studied. Results No differences were found among groups regarding RV echo‐Doppler and TDI variables. Sixteen significant correlations were found between RV TDI and left heart echocardiographic variables. Dogs with PH had significantly higher transmitral E wave peak velocity and higher E/eʹ ratio of septal (sMV) and lateral (pMV) mitral annulus. These 2 variables were found to predict presence of PH with a sensitivity of 84 and 72%, and a specificity of 71 and 80% at cut‐off values of 10 and 9.33 for sMV E/eʹ and pMV E/eʹ, respectively. Conclusions and clinical importance No association between variables of RV function and different MMVD stage and severity of PH could be detected. Some relationships were found between echocardiographic variables of right and left ventricular function.

Diagnostic accuracy of the vertebral heart score and other radiographic indices in the detection of cardiac enlargement in cats with different cardiac disorders

A retrospective search was conducted to evaluate the diagnostic accuracy of the vertebral heart score (VHS) and other related radiographic indices in the detection of cardiac enlargement associated with different cardiac disorders in the cat. One hundred and five cats with a complete echocardiographic examination and radiographic examination of the thorax with at least two orthogonal views were enrolled. Eighty-three cats had different cardiac disorders, 72 with left-sided cardiac disorders (LSCD) and 11 with right-sided cardiac disorders; 22 cats were free of cardiovascular abnormalities. Measurements of VHS and cardiac long and short axes on lateral (L) and dorsoventral or ventrodorsal radiographs were obtained. Receiver operating characteristic curves were calculated to evaluate the diagnostic accuracy of each radiographic index in differentiating between cats with cardiac disorders or cats with LSCD and cats without cardiac abnormalities and, among cats with LSCD, between those with no or mild left atrial enlargement (LAE) or those with moderate-to-severe LAE and healthy cats. The L-VHS at the cut-off of 7.9 had high diagnostic accuracy in distinguishing cats with LSCD and moderate-to-severe LAE from healthy cats, but all the other radiographic indices were moderately accurate in distinguishing between cats with overall cardiac disorders or LSCD, either with no or mild LAE and moderate-to-severe LAE, and healthy cats. The considered radiographic indices were also moderately accurate in predicting different degrees of LAE in cats with LSCD. Radiographic indices are reasonably specific, but less sensitive predictors of cardiac enlargement in cats with heart disorders.

The Vietnamese pig as a translational animal model to evaluate tissue engineered heart valves: promising early experience

Several animal models are currently used for the surgical implantation of either biologic or biopolymeric scaffolds in order to provide in vivo assessment of tissue-engineered heart valves. The Vietnamese pig (VP) is herein proposed as a suitable recipient to test the function of novel bioengineered valve substitutes, in the reconstruction of the right ventricular outflow tract (RVOT). This review aims to provide a complete and exhaustive panel of physiological parameters and methodological information for preclinical studies of tissue-engineered heart valves in the VP animal model.

Liver function evaluation in dogs by the galactose load test

The modern approach to hepatic liver disease in dogs can show hepatocellular damage, cholestasis and liver failure. Histology is the gold standard in specific disease diagnosis. In veterinary medicine there is no routine test for the quantitative evaluation of residual function or follow-up monitoring (Leville Webster, 2000). Dynamic tests have been introduced in human medicine: these consist of administration of substances metabolised in the liver and the subsequent study of their kinetics. In this way quantitative indications about specific hepatic metabolic functions are obtained (Brockmoller and Root, 1994). Some of these tests were much used in the past and have subsequently been neglected, but a recent revaluation is progressing in human (Beker, 1998) and veterinary medicine (Moeller et al., 2004). Galactose is a monosaccharide mainly metabolised by the hepatocyte. In humans the galactose elimination rate shows a direct relationship to functioning liver mass (Tygstrup, 1966). In dogs references are scarce and obsolete (Drill and Ivy, 1944): neither renal removal nor body distribution have been investigated (Figures 1 and 2). The aim of this work was to verify the reliability of the application of galactose load test in dogs, and to compare the concentration–time curves in healthy animals with those obtained in patients with liver diseases.

Microfilaricidal efficacy of a single administration of Advocate® (Bayer Animal Health) in dogs naturally infected with Dirofilaria immitis or Dirofilaria repens

The present study evaluated the microfilaricidal efficacy of a single application of the spot-on containing imidacloprid 10%/moxidectin 2.5% (Advocate(®), Bayer Animal Health) in dogs naturally infected either by Dirofilaria immitis or Dirofilaria repens. Dogs living in north-eastern and central-southern Italy, endemic for D. immitis and D. repens respectively, were randomly screened. Sixteen animals, eight infected with D. immitis and eight with D. repens, and fulfilling inclusion criteria were enrolled. Dogs infected with D. immitis received an adulticide treatment prior to the study and Advocate(®) 3 weeks after. The animals were divided in blocks of two (1:1, T1:T2) animals each, where Day 0 (D0) had an interval of 15days to compare T2 vs. T1 dogs during the first fortnight of examination (i.e. T2 dogs acted as control animals at each examination). At baseline (Days -15 and 0 for T2 and T1 dogs, respectively) the animals had a range of microfilaraemia of 180-99.700mff/ml (D. immitis) and 60-750 mff/ml (D. repens). All animals received a topical administration of Advocate(®) at D0 and were examined for microfilariae with microscopic and molecular tests at D15, D30, D60 and D90. All animals scored negative for mff at the first control post-treatment and throughout the study, with the exception of two D. immitis- infected animals that had a 2 mff/ml count at D15, and then become negative from Day 30 onwards. No adverse events were observed. The present study demonstrates the safety and the high microfilaricidal efficacy (99.97% and 100% for D. immitis and D. repens, respectively) of a single dose of moxidectin contained in Advocate(®) in naturally infected dogs.