Plasma atrial/A-type natriuretic peptide (ANP) concentration in horses with various heart diseases.

OBJECTIVE: Plasma atrial/A-type natriuretic peptide concentration (CpANP) was measured in horses presenting with various heart diseases to assess its potential diagnostic value. ANIMALS: Fifteen healthy horses (Group 1) and 60 horses with various heart diseases associated with normal chamber size and function (Group 2, n = 24), associated with abnormal left atrial (LA) size and/or function but normal left ventricle (LV) (Group 3, n = 19), or associated with both abnormal LA and LV size and/or function (Group 4, n = 17). METHODS: CpANP was measured by a commercially available radioimmunoassay. Echocardiographic measurements were compared between groups by one-way ANOVA and Holm-Sidak post-hoc test. Receiver operating characteristics (ROC) analyses were performed to identify the best cut-offs to distinguish between groups. Relations between echocardiographic measurements and biomarker concentrations were assessed with backward stepwise multiple linear regression. RESULTS: CpANP increased from Group 1 to 4 and was significantly higher in horses with heart disease than in controls. CpANP was associated with maximum LA area and LV fractional area change. The ROC analyses showed good specificity but poor sensitivity to distinguish between healthy horses and horses with heart disease overall, and between healthy horses and horses with altered left-sided chamber dimensions and/or function. CONCLUSION: CpANP is increased in horses with heart disease associated with altered left-sided chamber dimensions and/or function. However, its diagnostic value is compromised by poor sensitivity.

Atrial natriuretic peptide and cardiac troponin I concentrations in healthy Warmblood horses and in Warmblood horses with mitral regurgitation at rest and after exercise.

OBJECTIVE: Atrial natriuretic peptide (ANP) and cardiac troponin I (cTnI) serve as biomarkers for increased cardiac pressure/volume loading and for myocardial stress or damage. The objective was to describe the time course of plasma ANP concentrations (CpANP) and plasma cTnI concentrations (CpcTnI) in horses with mitral regurgitation (MR) compared to healthy horses at rest and after exercise, and to describe the relationship of CpANP with cardiac dimensions and intracardiac pressures. ANIMALS: 15 healthy Warmblood horses and 7 Warmblood horses with MR. METHODS: Cardiac dimensions at rest were measured using echocardiography. All horses underwent standardized treadmill exercise. Biomarker concentrations and intracardiac pressures were measured at rest and after exercise. Hypotheses were tested using statistical methods. The level of significance was P < 0.05. RESULTS: Horses with MR showed increased left atrial (LA) and left ventricular (LV) dimensions but similar exercise capacity compared to healthy horses. Pulmonary capillary wedge pressures (PCWP) and CpANP increased with exercise. Horses with MR had higher PCWP and higher CpANP at rest and after exercise compared to healthy horses, with the maximum difference in CpANP reached 10 min after exercise. CpANP was significantly related to PCWP and - although inconsistently and only in healthy horses - to echocardiographic indices of LA and LV size and function. CpcTnI was low throughout the study in both groups. CONCLUSIONS: CpANP is increased in horses with MR and is related to LA pressures and to left heart dimensions. MR is not necessarily associated with exercise intolerance and exercise-induced myocardial stress or damage.

Acute and chronic elevation of erythropoietin in the brain improves exercise performance in mice without inducing erythropoiesis.

Application of recombinant human erythropoietin (rhEpo) improves exercise capacity by stimulating red blood cell production that, in turn, enhances oxygen delivery and utilization. Apart from this, when applied at high doses, rhEpo crosses the blood-brain barrier, triggering protective neuronal effects. Here we show a fundamental new role by which the presence of Epo in the brain augments exercise performance without altering red blood cell production. Two different animal models, the transgenic mouse line Tg21, which constitutively overexpresses human Epo exclusively in the brain without affecting erythropoiesis, and wild-type mice treated with a single high dose of rhEpo, demonstrate an unexpected improvement in maximal exercise performance independent of changes in total hemoglobin mass, as well as in whole blood volume and cardiovascular parameters. This novel finding builds a more complete understanding regarding the central effects of endogenously produced and exogenously applied Epo on exercise performance.

Enhanced erythropoietin expression in the brainstem of newborn rats at high altitude.

In addition to its role in elevating red blood cell number, erythropoietin (Epo) exerts protective functions against acute and delayed degenerative diseases of the brain. Moreover, we have recently demonstrated that endogenously synthesized Epo and soluble Epo receptor (a negative regulator of Epo binding to the Epo receptor) in the central nervous system play a crucial role in facilitating the ventilatory response and acclimatization to hypoxia. Here we hypothesized that cerebral Epo in the brainstem is implicated in the process that allows cardiorespiratory acclimatization to high altitude hypoxia during the postnatal period. Thus, we evaluated the postnatal ontogeny of cerebral Epo concentration of Sprague-Dawley rats living and reproducing at high altitude for longer than 19 years (3600 m in La Paz, Bolivia). Our results show that postnatal Epo concentration in high-altitude rats is higher in the brainstem than in the forebrain. Moreover, although Epo concentration in the forebrain of high-altitude rats is similar to sea-level controls, Epo level in the brainstem is surprisingly 2-fold higher in high-altitude rats than in sea-level controls. These findings strongly suggest that brainstem Epo plays an important role in tolerance to high altitude hypoxia after birth. From a clinical perspective, a better understanding of the role of Epo in the postnatal development of cardiorespiratory responses in neonates exposed to acute or chronic hypoxia might be useful.

Quantifying Western blots: pitfalls of densitometry.

Although Western blots are frequently quantified, densitometry is not documented and appears to be based merely on traditions and guesswork. Confirming previous experience, none of 100 randomly selected and systematically scanned most recent papers provided sufficient information on how Western blot results were translated into statistical values. The importance of such information, however, becomes evident from our correlations of plasma erythropoietin values of various mammals determined using RIA and Western blot densitometry. Different common densitometry procedures applied to the identical Western blot revealed p-values of these correlations ranging from 0.000013 to 0.76 reflecting the necessity of a scientifically sound basis for densitometry of Western blots. At present, the current lack of any definitions in densitometry opens the door to uncontrollable acquisition of any desired p-value. Here we provide data that define what should be considered, what avoided and what documented when quantifying Western blots.

Inhibition of 4E-BP1 sensitizes U87 glioblastoma xenograft tumors to irradiation by decreasing hypoxia tolerance.

PURPOSE: Eukaryotic initiation factor 4E (eIF4E) is an essential rate-limiting factor for cap-dependent translation in eukaryotic cells. Elevated eIF4E activity is common in many human tumors and is associated with disease progression. The growth-promoting effects of eIF4E are in turn negatively regulated by 4E-BP1. However, although 4E-BP1 harbors anti-growth activity, its expression is paradoxically elevated in some tumors. The aim of this study was to investigate the functional role of 4E-BP1 in the context of solid tumors. METHODS AND MATERIALS: In vitro and in vivo growth properties, hypoxia tolerance, and response to radiation were assessed for HeLa and U87 cells, after stable expression of shRNA specific for 4E-BP1. RESULTS: We found that loss of 4E-BP1 expression did not significantly alter in vitro growth but did accelerate the growth of U87 tumor xenografts, consistent with the growth-promoting function of deregulated eIF4E. However, cells lacking 4E-BP1 were significantly more sensitive to hypoxia-induced cell death in vitro. Furthermore, 4E-BP1 knockdown cells produced tumors more sensitive to radiation because of a reduction in the viable fraction of radioresistant hypoxic cells. Decreased hypoxia tolerance in the 4E-BP1 knockdown tumors was evident by increased cleaved caspase-3 levels and was associated with a reduction in adenosine triphosphate (ATP). CONCLUSIONS: Our results suggest that although tumors often demonstrate increases in cap-dependent translation, regulation of this activity is required to facilitate energy conservation, hypoxia tolerance, and tumor radioresistance. Furthermore, we suggest that targeting translational control may be an effective way to target hypoxic cells and radioresistance in metabolically hyperactive tumors.

Erythropoietin protects from reperfusion-induced myocardial injury by enhancing coronary endothelial nitric oxide production.

OBJECTIVE: Cardioprotective properties of recombinant human Erythropoietin (rhEpo) have been shown in in vivo regional or ex vivo global models of ischemia-reperfusion (I/R) injury. The aim of this study was to characterize the cardioprotective potential of rhEPO in an in vivo experimental model of global I/R approximating the clinical cardiac surgical setting and to gain insights into the myocardial binding sites of rhEpo and the mechanism involved in its cardioprotective effect. METHODS: Hearts of donor Lewis rats were arrested with cold crystalloid cardioplegia and after 45 min of cold global ischemia grafted heterotopically into the abdomen of recipient Lewis rats. Recipients were randomly assigned to control non-treated or Epo-treated group receiving 5000 U/kg of rhEpo intravenously 20 min prior to reperfusion. At 5 time points 5-1440 min after reperfusion, the recipients (n=6-8 at each point) were sacrificed, blood and native and grafted hearts harvested for subsequent analysis. RESULTS: Treatment with rhEpo resulted in a significant reduction in myocardial I/R injury (plasma troponin T) in correlation with preservation of the myocardial redox state (reduced glutathione). The extent of apoptosis (activity of caspase 3 and caspase 9, TUNEL test) in our model was very modest and not significantly affected by rhEpo. Immunostaining of the heart tissue with anti-Epo antibodies showed an exclusive binding of rhEpo to the coronary endothelium with no binding of rhEpo to cardiomyocytes. Administration of rhEpo resulted in a significant increase in nitric oxide (NO) production assessed by plasma nitrite levels. Immunostaining of heart tissue with anti-phospho-eNOS antibodies showed that after binding to the coronary endothelium, rhEpo increased the phosphorylation and thus activation of endothelial nitric oxide synthase (eNOS) in coronary vessels. There was no activation of eNOS in cardiomyocytes. CONCLUSIONS: Intravenous administration of rhEpo protects the heart against cold global I/R. Apoptosis does not seem to play a major role in the process of tissue injury in this model. After binding to the coronary endothelium, rhEpo enhances NO production by phosphorylation and thus activation of eNOS in coronary vessels. Our results suggest that cardioprotective properties of rhEpo are at least partially mediated by NO released by the coronary endothelium.

Placental HIFs as markers of cerebral hypoxic distress in fetal mice.

Reduced oxygen supply during the pre- and perinatal period often leads to acquired neonatal brain damage. So far, there are no reliable markers available to assess the hypoxic cerebral damage and the resulting prognosis during the immediate postnatal period. Thus we aimed to determine whether the hypoxia-inducible transcription factors (HIF-1 and HIF-2) and/or their target genes in the placenta represent reliable indicators of hypoxic distress of the developing brain during systemic hypoxia at the end of gestation. To this end, pregnant mice were exposed to systemic hypoxia (inspired O2 fraction: 6%, 6 h) at gestational day 20. This hypoxic exposure significantly increased HIF-1alpha and HIF-2alpha protein levels in brain and placental tissue. Compared with normoxic controls, an increase of HIF-1alpha-immunoreactive neurons and HIF-2alpha-positive glial cells and vascular endothelial cells was observed in hypoxic cerebral cortex and hippocampus. In placenta, HIF-1alpha and HIF-2alpha were expressed in labyrinthine layer with increased staining intensity during hypoxia compared with normoxia. Significant upregulation of VEGF mRNA and protein in brain and placenta, as well as erythropoietin protein in placenta, indicated activity of the HIF system upon fetal hypoxia. Notably, hypoxia did not affect expression of the HIF target genes inducible nitric oxide synthase and GLUT-1. Taken together, at gestational day 20, systemic hypoxia led to upregulation of HIF-alpha in mouse brain that was temporally paralleled in placenta, implying that alpha-subunits of both HIF-1 and HIF-2 are indeed early markers of hypoxic distress in vivo. If our data reflect the situation in humans, analysis of the placenta will allow early identification of the hypoxic brain distress occurring near birth.

Abortion in mice with excessive erythrocytosis is due to impaired arteriogenesis of the uterine arcade.

We postulate that repeated pregnancy loss, intrauterine growth restriction, and preeclampsia are caused by impaired elevation of uterine blood flow due to disturbed arteriogenesis of the uterine arcade. This hypothesis is based on the observation that pregnant human erythropoietin-overexpressing (plasma levels elevated 12-fold) mice (termed tg6 mice) suffering from excessive erythrocytosis generally abort at midgestation unless their hematocrit of 0.85 is drastically lowered. Transgenic mice show placental malformations that parallel those observed in pregnant women suffering from impaired uterine perfusion. Shear stress, a key factor inducing arteriogenesis, was 5-fold lower in tg6 mice compared with wildtype (WT) littermates. Consequently, uterine artery growth was reduced, and dramatically fewer viable pups (1.63 +/- 2.20 vs. 8.10 +/- 0.74 in WT) of lower weight (1.29 +/- 0.07 g vs. 1.62 +/- 0.12 g in WT) were delivered in first pregnancies. Only in subsequent pregnancies did tg6 deliver approximately the expected number of pups. Birth weights of tg6 offspring, however, remained reduced. As the spleen is a major site of extramedullary erythropoiesis in tg6 animals, splenectomy reduced the hematocrit to 0.6-0.7. In turn, shear stress increased to normal values, and splenectomized primiparous tg6 showed normal uterine artery growth and delivery of pups similar in number and weight compared with WT. We conclude that poor arteriogenesis is a previously unappreciated cause for clinically important pregnancy complications.

Analytical validation of commercial immunoassays for the measurement of cardiovascular peptides in the dog.

Immunoassays for the measurement of concentrations of the cardiovascular peptides pro-atrial natriuretic peptide (proANP), brain natriuretic peptide (BNPPen and BNPPhoe), endothelin-1 (ET-1Bio, ET-1IBL and ET-1Phoe) and big endothelin-1 (Big-ETBio and Big-ETIBL) were validated in canine serum by determination of intra-assay variability and dilutional parallelism. Commercial kits that showed good results were further validated by determination of intra- and inter-assay variability, dilutional parallelism and spiking recovery. Assays for proANP, BNPPhoe, ET-1IBL and Big-ETIBL showed acceptable results in the preliminary validation and were fully validated. The intra- and inter-assay variability was acceptable for all four assays, linearity was demonstrated and recovery rates were acceptable. The performances of the different immunoassays varied considerably, underscoring the importance of validation. Of the assays studied, proANP, BNP(Phoe), ET-1IBL and Big-ETIBL produced precise, reproducible and accurate results and can be recommended for clinical application.

Constitutively overexpressed erythropoietin reduces infarct size in a mouse model of permanent coronary artery ligation.

In view of the emerging role of recombinant human erythropoietin (rhEPO) as a novel therapeutical approach in myocardial ischemia, we performed the first two-way parallel comparison to test the effects of rhEPO pretreatment (1000 U/kg, 12h before surgery) versus EPO transgenic overexpression in a mouse model of myocardial infarction. Unlike EPO transgenic mice who doubled their hematocrit, rhEPO pretreated mice maintained an unaltered hematocrit, thereby offering the possibility to discern erythropoietic-dependent from erythropoietic-independent protective effects of EPO. Animals pretreated with rhEPO as well as EPO transgenic mice underwent permanent left anterior descending (LAD) coronary artery ligation. Resulting infarct size was determined 24h after LAD ligation by hematoxylin/eosin staining, and morphometrical analysis was performed by computerized planimetry. A large reduction in infarction size was observed in rhEPO-treated mice (-74% +/- 14.51; P = 0.0002) and an even more pronounced reduction in the EPO transgenic group (-87% +/- 6.31; P < 0.0001) when compared to wild-type controls. Moreover, while searching for novel early ischemic markers, we analyzed expression of hypoxia-sensitive Wilms' tumor suppressor gene (WT1) in infarcted hearts. We found that its expression correlated with the infarct area, thereby providing the first demonstration that WT1 is a useful early marker of myocardial infarction. This study demonstrates for the first time that, despite high hematocrit levels, endogenously overexpressed EPO provides protection against myocardial infarction in a murine model of permanent LAD ligation.

Interrelation of directly measured oxygenation levels, erythropoietin and erythropoietin receptor expression in spontaneous canine tumours.

The expression of the hypoxia-inducible protein erythropoietin in tumour cells correlates with levels of tumour hypoxia. Our aim was to look for an interrelation of directly measured oxygenation levels, the presence of tissue erythropoietin and its receptor. Data of tumour oxygenation status, plasma and tissue erythropoietin and its receptor in a group of spontaneously occurring tumours in 15 dogs were collected. Polarographic tumour oxygen partial pressure measurements were obtained and data were correlated. Significant positive correlations were found between tissue erythropoietin and the percentages of pO2 values < or = 10 mmHg. Multivariate analysis revealed no parameters influencing plasma erythropoietin levels. Our results show that a co-expression of erythropoietin receptor and its ligand in spontaneous canine tumours exists, that the level of hypoxia in tumour cells correlates with the level of tissue erythropoietin and suggest the need to be quantitatively and functionally tested as novel prognostic biological parameters in neoplastic tissues.

High altitude training of dogs results in elevated erythropoietin and endothelin-1 serum levels.

Living at 2300-m altitude combined with intermittent training at 3500 m leads to cardiovascular alterations in dogs, including increase in systemic and pulmonary artery pressure. Despite moderate to marked hypoxemia at these altitudes, erythrocytosis does not develop. To study humoral mechanisms of acclimatisation to high altitude, erythropoietin (EPO), endothelin-1 (ET-1), big endothelin (Big-ET) and vascular endothelial growth factor (VEGF) were measured in dogs living at 2300 m and intermittently ascending to 3500 m, and compared to the values obtained in control dogs living at 700-900 m. While the median EPO and ET-1 level in dogs at 2300 m did not differ from the one measured at 700-900 m, exposure from 2300 to 3500 m resulted in significantly elevated EPO and ET-1 levels. Big-ET levels were significantly higher at 2300 and 3500 m compared to dogs at low altitude, but did not differ between 2300 and 3500 m. VEGF was significantly elevated in dogs at 2300 m compared to dogs at low altitude. The increases in EPO, VEGF, ET-1 and Big-ET are thought to reflect the effect of hypoxia on a cellular level in these dogs. Obviously, the mild elevation of EPO levels observed at 3500 m was not sufficient to cause erythrocytosis. Elevations of the vasoconstrictors Big-ET and ET-1 may play some, but not a central role in hypoxic vasoconstriction in these dogs. Finally, serum VEGF measurement may be a sensitive and useful test to assess hypoxic stress in dogs.