Contrasting effects of transforming growth factor ß1 on programmed cell death of bovine mammary epithelial cell lines MAC-T and BME-UV1.

A previous study in the bovine mammary epithelial cell line BME-UV1 demonstrated that suppression of the phosphatidylinositol-4,5-biphosphate 3 kinase (PI3K)/AKT (somatotropic) signaling pathway was required for transforming growth factor ß1 (TGFß1)-induced programmed cell death (PCD). To investigate whether this is a universal mechanism for TGFß1 to induce PCD in bovine mammary epithelium, we compared TGFß1 modulation of PI3K/AKT and its role in PCD in 2 bovine mammary epithelial cell lines: MAC-T and BME-UV1. In MAC-T cells, TGFß1 promoted cell survival, and this paralleled a reduction in PI3K/AKT activity, rather than an increase. In BME-UV1 cells, TGFß1 induced PCD, and this was accompanied by a time-dependent effect on PI3K/AKT activity, including an initial significant increase in the phosphorylation of AKT at 3 h, followed by a reduction between 12 and 24 h, and then an increase at 48 h. Inhibition of AKT activity enhanced TGFß1-induced PCD in BME-UV1 cells but had no effect on MAC-T cells, suggesting that TGFß1 mediates PCD in BME-UV1 cells through suppression of AKT activity. Inhibition of TGFß receptor type I (TßRI) kinase activity completely abrogated TGFß1-induced PCD in BME-UV1 cells but had no effect on TGFß1-induced suppression of PCD in MAC-T cells, demonstrating that TGFß1-induced PCD in BME-UV1 cells is dependent on TßRI/SMAD signaling. These and previous observations suggest that the different effects of TGFß1 on PCD in these cell lines might involve noncanonical signaling pathways other than PI3K/AKT, and may reflect their different lineages. Future studies should address this finding, taking into consideration the effect that different culture conditions might have on cell phenotype.

Melatonin and IL-25 modulate apoptosis and angiogenesis mediators in metastatic (CF-41) and non-metastatic (CMT-U229) canine mammary tumour cells.

BACKGROUND: Melatonin has oncostatic actions and IL-25 is active in inflammatory processes that induce apoptosis in tumor cells AIM: The aim of this study was to evaluate melatonin and IL-25 in metastatic (CF-41) and non-metastatic (CMT-U229) canine mammary tumor cells cultured as monolayers and tridimensional structures. MATERIALS AND METHODS: The cells were treated with melatonin, IL-25 and IL-17B silencing gene and performed cell viability, gene and protein expression of caspase-3 and VEGFA (Vascular endothelial growth factor A) and an apoptosis membrane protein array. RESULTS: Treatment with 1 mM of melatonin reduced cell viability of both tumor cell lines, all treatments alone and combined significantly increased caspase-3 cleaved and proteins involved in the apoptotic pathway and reduced pro-angiogenic VEGFA, confirming the effectiveness of these potential promising treatments. CONCLUSION: This is the first study evaluating the potential use of these strategies in CF-41 and CMT-U229 cell lines and together encourages subsequent in vitro and in vivo studies for further exploration of clinical applications.

Release of endothelial cell associated VEGFR2 during TGF-ß modulated angiogenesis in vitro.

BACKGROUND: Sprouting angiogenesis requires vascular endothelial proliferation, migration and morphogenesis. The process is regulated by soluble factors, principally vascular endothelial growth factor (VEGF), and via bidirectional signaling through the Jagged/Notch system, leading to assignment of tip cell and stalk cell identity. The cytokine transforming growth factor beta (TGF-ß) can either stimulate or inhibit angiogenesis via its differential surface receptor signaling. Here we evaluate changes in expression of angiogenic signaling receptors when bovine aortic endothelial cells were exposed to TGF-ß1 under low serum conditions. RESULTS: TGF-ß1 induced a dose dependent inhibition of tip cell assignment and subsequent angiogenesis on Matrigel, maximal at 5.0 ng/ml. This occurred via ALK5-dependent pathways and was accompanied by significant upregulation of the TGF-ß co-receptor endoglin, and SMAD2 phosphorylation, but no alteration in Smad1/5 activation. TGF-ß1 also induced ALK5-dependent downregulation of Notch1 but not of its ligand delta-like ligand 4. Cell associated VEGFR2 (but not VEGFR1) was significantly downregulated and accompanied by reciprocal upregulation of VEGFR2 in conditioned medium. Quantitative polymerase chain reaction analysis revealed that this soluble VEGFR2 was not generated by a selective shift in mRNA isoform transcription. This VEGFR2 in conditioned medium was full-length protein and was associated with increased soluble HSP-90, consistent with a possible shedding of microvesicles/exosomes. CONCLUSIONS: Taken together, our results suggest that endothelial cells exposed to TGF-ß1 lose both tip and stalk cell identity, possibly mediated by loss of VEGFR2 signaling. The role of these events in physiological and pathological angiogenesis requires further investigation.

Antemortem diagnosis of a left auricular appendage herniation through a partial pericardial defect in a dog with degenerative mitral valve disease.

INTRODUCTION: A 14-year-old neutered male crossbreed dog was presented for weakness, cough and weight loss. Cardiac auscultation revealed tachycardia, arrhythmia and a grade V/VI left apical systolic heart murmur. Thoracic radiographs showed a large homogeneous soft tissue opacity in close contact with the cardiac silhouette in the left cranioventral mediastinum. Cardiac evaluation showed atrial fibrillation, degenerative mitral valve disease and a dilated left auricular appendage outside the pericardium consistent with herniation through a partial pericardial defect. Seven months after diagnosis, an atrial septal defect secondary to acquired atrial septal rupture was identified. The dog was euthanized thirteen months after initial presentation because of unresponsive clinical signs of congestive heart failure.

Direct assignment of molecular vibrations via normal mode analysis of the neutron dynamic pair distribution function technique.

For over a century, vibrational spectroscopy has enhanced the study of materials. Yet, assignment of particular molecular motions to vibrational excitations has relied on indirect methods. Here, we demonstrate that applying group theoretical methods to the dynamic pair distribution function analysis of neutron scattering data provides direct access to the individual atomic displacements responsible for these excitations. Applied to the molecule-based frustrated magnet with a potential magnetic valence-bond state, LiZn2Mo3O8, this approach allows direct assignment of the constrained rotational mode of Mo3O13 clusters and internal modes of MoO6 polyhedra. We anticipate that coupling this well known data analysis technique with dynamic pair distribution function analysis will have broad application in connecting structural dynamics to physical properties in a wide range of molecular and solid state systems.

Systolic arterial blood pressure in small-breed dogs with degenerative mitral valve disease: a prospective study of 103 cases (2007-2012).

The objective of this prospective observational study was to assess systolic arterial blood pressure (SABP) in small-breed dogs with degenerative mitral valve disease (MVD) from different International Small Animal Cardiac Health Council (ISACHC) heart failure classes. For this purpose, 103 client-owned dogs weighing <20 kg (mean ± standard deviation, 8.5 ± 3.0 kg; aged 9.8 ± 2.9 years) and presenting with MVD diagnosed by echo-Doppler examination were enrolled. Nineteen healthy dogs (9.9 ± 2.3 years; 8.7 ± 4.2 kg) were concurrently recruited as controls. SABP was measured in unsedated dogs using the Doppler method according to the recommendations in the American College of Veterinary Medicine consensus statement. SABP was significantly increased in dogs in ISACHC class 1 (n=53; median, interquartile range 140 mmHg, 130-150 mmHg) and class 2 (n=21; 140 mmHg, 130-150 mmHg), compared to the control group (n=19; 130 mmHg, 120-140 mmHg; P<0.01 and P<0.05, respectively), but remained within the reference interval (≤ 160 mmHg). Conversely, dogs in ISACHC class 3 showed a significantly lower SABP (n=29, 120 mmHg, 110-130 mmHg) than those from all other ISACHC classes (P<0.001) and the controls (P<0.05). Additionally, SABP<120 mmHg was recorded in 13/103 dogs (13%). The 13 dogs were all ISACHC class 3 (3a or 3b) and were under medical treatment for heart failure. In conclusion, MVD was often associated with SABP values that were within the reference interval, but at its upper end. However, a significant decrease in SABP was observed in dogs with ISACHC heart failure class 3. Whether such low SABP values resulted from an MVD-related decrease in cardiac output, an afterload reduction owing to cardiac treatment, or both, remains to be determined.

Comparative echocardiographic and clinical features of hypertrophic cardiomyopathy in 5 breeds of cats: a retrospective analysis of 344 cases (2001-2011).

BACKGROUND: Primary hypertrophic cardiomyopathy (HCM) is the most common feline heart disease and has been demonstrated to be inherited in some breeds. However, few studies have compared HCM phenotypes and survival according to breed. OBJECTIVES: To compare epidemiological characteristics, clinical findings, left ventricular (LV) geometric patterns, and survival in several breeds of cats with HCM. ANIMALS: Three hundred and forty-four cats from 5 different breeds (Persian, Domestic Shorthair [DS], Sphynx, Maine coon [MC], and Chartreux) with primary HCM diagnosed by conventional echocardiography. METHODS: Retrospective study. Cats were classified according to breed and clinical status. RESULTS: Age at the time of diagnosis was lower (P < .001) in MC (median age, 2.5 years) and Sphynx (3.5 years) than in other breeds (OB), ie, 8.0, 8.0, and 11.0 years for DS, Chartreux, and Persians, respectively. The prevalence of LV outflow tract obstruction was higher (P < .001) in Persians (23/41; 56%) than in OB (115/303; 38%). Age at the first cardiac event was lower (P < .01) in MC (median age, 2.5 years) than in OB (7.0 years). All cats surviving > 15 years of age were DS, Persians, or Chartreux. Sudden death (representing 24% of all cardiac deaths) was observed only in 3 breeds (DS, MC, and Sphynx). CONCLUSION AND CLINICAL IMPORTANCE: As in humans, feline HCM is characterized by marked phenotypic variability with several breed-dependent features regarding epidemiology, LV geometric patterns, and clinical course (ie, age at diagnosis, 1st cardiac event, and cause of death).

Echocardiographic and tissue Doppler imaging alterations associated with spontaneous canine systemic hypertension.

BACKGROUND: Feline systemic arterial hypertension (SHT) is associated with a wide spectrum of left ventricular (LV) geometric patterns as well as diastolic, and to a lesser extent, systolic myocardial dysfunction. However, little is known about SHT-related cardiac changes in dogs. HYPOTHESIS: SHT in dogs is responsible for morphological and functional cardiac alterations. ANIMALS: Thirty dogs with spontaneous untreated SHT and 28 age- and body weight-matched healthy dogs as controls. METHODS: Prospective observational study. Conventional echocardiography and 2-dimensional color tissue Doppler imaging were performed in SHT dogs by trained observers and compared with controls. RESULTS: Forty-seven percent of SHT dogs (14/30) had diffuse concentric hypertrophy. None had left atrial dilatation and 10/30 (33%) had aortic insufficiency (AoI) associated with proximal aortic dilatation. Longitudinal diastolic left ventricular free wall (LVFW) motion was altered in all SHT dogs at the base (early to late diastolic wave ratio, E/A = 0.5 ± 0.1 versus 1.3 ± 0.3 for controls, P < .0001) and the apex (E/A = 1.6 ± 1.7 versus 3.9 ± 3.1, P < .05). Longitudinal motion of the interventricular septum at the base (E/A = 0.7 ± 0.4 versus 1.1 ± 0.1, P < .01) and radial LVFW motion in the subendocardium (E/A = 0.9 ± 0.5 versus 1.6 ± 0.3, P < .01) were also altered in dogs with SHT. Longitudinal LVFW systolic velocities and gradients were also significantly decreased (P < .05) in SHT dogs. CONCLUSION AND CLINICAL IMPORTANCE: As in SHT in cats, SHT in dogs is associated with myocardial dysfunction independently of the presence of myocardial hypertrophy. However, unlike feline SHT, it results in a homogeneous LV geometric pattern with a relatively high prevalence of AoI.

Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors.

The overexpression in tumor cells of (proto)-oncogenic receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) or ErbB2/neu (also known as HER-2) is generally thought to contribute to the development of solid tumors primarily through their effects on promoting uncontrolled cell proliferation. However, agents that antagonize the function of the protein products encoded by these (proto)-oncogenes are known to behave in vivo in a cytotoxic-like manner. This implies that such oncogenes may regulate critical cell survival functions, including angiogenesis. The latter could occur as a consequence of regulation of relevant growth factors by such oncogenes. We therefore sought to determine whether EGFR or ErbB2/neu may contribute to tumor angiogenesis by examining their effects on the expression of vascular endothelial cell growth factor (VEGF)/vascular permeability factor (VPF), one of the most important of all known inducers of tumor angiogenesis. We found that in vitro treatment of EGFR-positive A431 human epidermoid carcinoma cells, which are known to be heavily dependent on VEGF/VPF in vivo as an angiogenesis growth factor, with the C225 anti-EGFR neutralizing antibody caused a dose-dependent inhibition of VEGF protein expression. Prominent suppression of VEGF/VPF expression in vivo, as well as a significant reduction in tumor blood vessel counts, were also observed in established A431 tumors shortly after injection of the antibody as few as four times into nude mice. Transformation of NIH 3T3 fibroblasts with mutant ErbB2/neu, another EGFR-like oncogenic tyrosine kinase, resulted in a significant induction of VEGF/VPF, and the magnitude of this effect was further elevated by hypoxia. Moreover, treatment of ErbB2/neu-positive SKBR-3 human breast cancer cells in vitro with a specific neutralizing anti-ErbB2/neu monoclonal antibody (4D5) resulted in a dose-dependent reduction of VEGF/VPF protein expression. Taken together, the results suggest that oncogenic properties of EGFR and ErbB2/neu may, at least in part, be mediated by stimulation of tumor angiogenesis by up-regulating potent angiogenesis growth factors such as VEGF/VPF. These genetic changes may cooperate with epigenetic/environmental effects such as hypoxia to maximally stimulate VEGF/VPF expression. Therapeutic disruption of EGFR or ErbB2/neu protein function in vivo may therefore result in partial suppression of angiogenesis, a feature that could enhance the therapeutic index of such agents in vivo and endow them with anti-tumor effects, the magnitude of which may be out of proportion with their observed cytostatic effects in monolayer tissue culture.

Development of an ELISA to study the polymorphism of dextromethorphan oxidation in a French population.

Simple, sensitive and selective enzyme immunoassays (ELISA) for monitoring urinary dextromethorphan and its major metabolite, dextrorphan, were developed. Dextromethorphan and dextrorphan hemisuccinates were linked to bovine serum albumin and specific antisera against each immunogen were raised in rabbits. The sensitivity of the ELISA was high (limit of detection 740 and 600 pg.ml-1 for dextromethorphan and dextrorphan, respectively). Intra- and interassay variation was less than 10%, and cross-reactivity between the two compounds was less than 1%. The ELISA was employed to phenotype 216 French subjects. The frequency of poor metabolizers was 5.1%.