Chronic Cadmium Exposure Accelerates the Development of Atherosclerosis and Induces Vascular Dysfunction in the Aorta of ApoE-/- Mice.

Cadmium exposure is related to cardiovascular diseases, including hypertension, atherosclerosis, increased oxidative stress, endothelial dysfunction, and specific biochemical changes induced by this metal. Thus, we aimed to investigate whether cadmium exposure induces endothelial dysfunction, accelerates atherosclerotic plaque formation in the aorta, and enhances oxidative stress in apolipoprotein E knockout (ApoE-/-) mice. Experiments were performed in 14-week-old male wild-type and ApoE-/- mice. ApoE-/- mice received cadmium (CdCl2 100 mg/L in drinking water for 28 days) or vehicle (distilled water). After treatment, vascular reactivity to phenylephrine, acetylcholine, and sodium nitroprusside was analyzed using isolated aorta. Bone marrow cells were isolated to assess the production of nitric oxide and reactive oxygen and nitrogen species. ApoE-/- cadmium-treated mice had higher cholesterol levels than non-exposed mice. Cadmium exposure decreased the vasodilatation response to acetylcholine in aortic ring of ApoE-/- mice, though no changes in phenylephrine or sodium nitroprusside responses were observed. L-NAME reduced vasodilator responses to acetylcholine; this effect was lower in ApoE-/- cadmium-treated mice, suggesting reduction in nitric oxide (NO) bioavailability. Moreover, in bone marrow cells, cadmium decreased cytoplasmic levels of NO and increased superoxide anions, hydrogen peroxide, and peroxynitrite in ApoE-/- mice. Morphological analysis showed that cadmium exposure increased plaque deposition in the aorta by approximately 3-fold. Our results suggest that cadmium exposure induces endothelial dysfunction in ApoE-/- mice. Moreover, cadmium increased total cholesterol levels, which may promote the early development of atherosclerosis in the aorta of ApoE-/- mice. Our findings support the hypothesis that cadmium exposure might increase the risk of atherosclerosis.

Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region.

Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

Reference range of gestational uterine artery resistance index in small canine breeds.

The objectives of this study were to evaluate the uterine artery blood flow during the second half of normal pregnancy in small breed dogs and to determine the RI reference values of this artery in these bitches. Fifty-two, 1.5-6.5 kg, purebred healthy pregnant bitches were included in this study. Doppler ultrasonographic examinations of the uterus were performed every 10 days from Day 30 of the estrous cycle to parturition. Peak systolic velocity (PSV) and end diastolic velocity (EDV) of the uterine artery were measured while RI [(PSV-EDV)/PSV] was automatically calculated. Values of PSV, EDV and RI were analyzed by repeated measures ANOVA followed by Tukey test using age, body weight, litter size and parity as covariates. Resistance index reference ranges were constructed by regressing this index on gestational age. Equation of the polynomial regression curves was used to calculate the mean, 95th and 5th centiles for each gestational age (GA). A gradual increase of PSV (P < 0.01) and EDV (P < 0.01) and a decrease in RI (P < 0.01) were found throughout the study period. PSV and EDV were influenced by litter size (P < 0.05), while RI was not. None of the Doppler parameters were influenced by age, body weight or parity. A second-degree polynomial described the relationship between RI of uterine artery and GA in the different time points: [RI = 0.993 + 8.4x10-5 x GA2- 0.0117 x GA]. It is concluded that uterine artery blood flow progressively increased throughout normal pregnancy in small breed bitches. In addition, reference ranges of uterine artery RI were 0.64-0.79, 0.57 to 0.75, 0.53 to 0.71 and 0.52 to 0.66 on Days 30, 40, 50 and 60 of gestation, respectively.

Echocardiographic and electrocardiographic parameters during the normal postpartum period in toy breeds of dogs.

The aim of this study was to evaluate echocardiographic and electrocardiographic parameters during the normal canine postpartum period. Twenty clinically healthy pregnant bitches of toy breeds (11 Miniature poodles, five Yorkshire terriers, two Maltese terriers and two Bichons Frises) were evaluated on days -3, 3, 10, 17, 24, 38, 52 and 80 relative to parturition (day 0). During the first postpartum week, the width of the interventricular septum in systole, the shortening fraction and the left atrium size decreased, while the left ventricle internal diameter in systole and end systolic stress increased. There were progressive decreases in the velocity of circumferential fibre shortening, stroke volume, cardiac output, and mitral E and A wave values. Systolic blood pressure increased markedly during the first postpartum week to gradually increase thereafter. Heart rate and corrected QT interval progressively decreased, while P wave amplitude increased. QRS complex amplitude decreased in the second week after parturition and then increased during the following weeks. In conclusion, there were changes in systolic function and some structural adaptive changes in the bitch during the first 80days postpartum. In addition, maternal heart rate and corrected QT interval decreased, while P wave and QRS amplitudes increased.

Uterine blood flow evaluation in bitches suffering from cystic endometrial hyperplasia (CEH) and CEH-pyometra complex.

Doppler ultrasound is a useful diagnosis tool to evaluate uterine blood flow in different canine reproductive states. The aim of this study was to describe and compare uterine blood flow in bitches suffering from cystic endometrial hyperplasia (CEH) and CEH-pyometra complex (CEH-P). Ninety diestrous bitches were clinically, hematologically, and ultrasonographically classified into four groups: (1) clinical signs, leukocytosis, CEH, and uterine luminal contents (CEH-P, n = 31); (2) asymptomatic bitches with CEH and uterine contents (CEH-C, n = 15); (3) asymptomatic bitches with CEH without uterine contents (CEH, n = 16); and (4) normal diestrous bitches (ND, n = 28). The widest cross-sectional diameter and uterine wall thickness (W) of uterine horns were measured using two-dimensional ultrasound. Peak systolic velocity and end diastolic velocity of uterine arteries were measured by Doppler ultrasound in all the bitches. Peak systolic velocity and end diastolic velocity were higher in CEH-P than in CEH-C, CEH, and ND (P < 0.01); although both parameters did not show differences among the latter three groups (P > 0.1). Conversely, resistance index (RI) reported lower values in CEH-P than that in the other three groups (P < 0.01), being ND higher (P < 0.01) than both CEH-C and CEH which did not differ between themselves (P > 0.1). When all the bitches were considered, correlation between RI and cross-sectional diameter was r = -0.69 (P < 0.01) and between RI and uterine wall thickness was r = -0.02 (P > 0.1). It is concluded that the uterine artery blood flow velocity of bitches suffering from pyometra was higher, not only from normal bitches, but also from females with endometrial hyperplasia. Furthermore, endometrial hyperplasia, accompanied or not by luminal contents, had a higher blood flow velocity than normal uterus. Hemodynamic parameters appear as useful markers to differentiate uterine pathologic conditions.

Bidimensional and Doppler ultrasonographic evaluation of postpartum uterine involution in the queen.

The aim of this study was to describe bidimensional and Doppler ultrasonographic changes of uterine involution during normal feline puerperium. Secondary, the postpartum vaginal discharge was described. Twelve pregnant female cats were included in this study. After queening, vulvar discharge was grossly and microscopically examined daily. Bidimensional and Doppler ultrasonographic examinations of the uterus were performed on Days -4 to -2, 4, 11, 18, and 25 from parturition. Total uterine diameter, uterine wall thickness, uterine lumen contents, peak systolic velocity, end diastolic velocity, and resistance index of uterine arteries were measured. The cats presented serosanguineous vulvar discharge for a mean of 3 ± 1 days after parturition, and the cytology revealed 70% to 80% of erythrocytes, which progressively decreased up to Day 13. Immediately after parturition, there were less than 20% neutrophils, and this percentage gradually diminished to 0% to 1% at the end of the study. Uterine total diameter diminished up to Day 25 (P < 0.01), when ultrasonographic uterine dimensions were similar to that of anestrus. A progressive decrease of uterine wall thickness (P < 0.05), uterine lumen contents (P < 0.01), peak systolic velocity (P < 0.01), and end diastolic velocity (P < 0.01) was found throughout the study period. Conversely, resistance index increased during the first week after parturition (P < 0.01). It is concluded that the uterine artery blood flow progressively decreased during the first 25 days after parturition, which was associated with the bidimensional ultrasonographic regression of the organ. Although lochial discharge disappeared far before ultrasonographic involution, cytologic findings further corroborated the duration of this regression process.

Toll-like receptor 4 contributes to vascular remodelling and endothelial dysfunction in angiotensin II-induced hypertension.

BACKGROUND AND PURPOSE: Toll-like receptor 4 (TLR4) signalling contributes to inflammatory cardiovascular diseases, but its role in hypertension and the associated vascular damage is not known. We investigated whether TLR4 activation contributed to angiotensin II (AngII)-induced hypertension and the associated vascular structural, mechanical and functional alterations. EXPERIMENTAL APPROACH: AngII was infused (1.44 mg · kg(-1) · day(-1), s.c.) for 2 weeks in C57BL6 mice, treated with a neutralizing anti-TLR4 antibody or IgG (1 µg · day(-1); systolic BP (SBP) and aortic cytokine levels were measured. Structural, mechanical and contractile properties of aortic and mesenteric arterial segments were measured with myography and histology. RT-PCR and Western blotting were used to analyse these tissues and cultured vascular smooth muscle cells (VSMC) from hypertensive rats (SHR). KEY RESULTS: Aortic TLR4 mRNA levels were raised by AngII infusion. Anti-TLR4 antibody treatment of AngII-treated mice normalised: (i) increased SBP and TNF-α, IL-6 and CCL2 levels; (ii) vascular structural and mechanical changes; (iii) altered aortic phenylephrine- and ACh-induced responses; (iv) increased NOX-1 mRNA levels, superoxide anion production and NAD(P)H oxidase activity and effects of catalase, apocynin, ML-171 and Mito-TEMPO on vascular responses; and (v) reduced NO release and effects of L-NAME on phenylephrine-induced contraction. In VSMC, the MyD88 inhibitor ST-2825 reduced AngII-induced NAD(P)H oxidase activity. The TLR4 inhibitor CLI-095 reduced AngII-induced increased phospho-JNK1/2 and p65 NF-κB subunit nuclear protein expression. CONCLUSIONS AND IMPLICATIONS: TLR4 up-regulation by AngII contributed to the inflammation, endothelial dysfunction, vascular remodelling and stiffness associated with hypertension by mechanisms involving oxidative stress. MyD88-dependent activation and JNK/NF-κB signalling pathways participated in these alterations.

Doppler ultrasonographic assessment of uterine arteries during normal canine puerperium.

The aim of this study was to describe Doppler ultrasonographic changes of uterine arteries during normal canine puerperium. Eight healthy, pure-bred bitches, were ultrasonographically assessed during the postpartum period on Days -3, 3, 10, 17, 24, 38, 52 and 80 (Day 0 defined as the day of parturition). Total horn diameters (TD) and endometrium thickness (E) were evaluated. Color Doppler was used to localize uterine arteries at both sides of the body and pulsed-wave Doppler was performed to obtain the waveforms. Peak systolic velocity (PSV) and end diastolic velocity (EDV) were measured. Resistance index [(PSV-EDV)/PSV] was automatically calculated. Values of TD, E, PSV, EDV and RI were analyzed by repeated measures ANOVA followed by LSD test (SPSS 18.0; SPSS, Chicago, IL, USA). A correlation analysis was also carried out between RI and TD. A progressive decrease of TD (P<0.01) and E (P<0.01) was found in the course of the study. A gradual diminution of PSV (P<0.01) and EDV (P<0.01) and an increase of RI (P<0.01) were also found throughout the study period. The resistance index negatively correlated with TD (r=-0.46; P<0.01) and E (r=-0.44; P<0.01) while the ultrasonographic and vascular changes in this period are concurrent with regenerative changes in the glandular and epithelial structures of the uterus. It is concluded that uterine artery RI progressively increased during normal canine puerperium, associated to the two-dimensional ultrasonographic regression of the organ.

Deleterious effect of hypothermia in myocardial protection against cold ischemia: a comparative study in isolated rat hearts.

BACKGROUND: There is a growing need to improve heart preservation benefit the performance of cardiac operations, decrease morbidity, and more important, increase the donor pool. Therefore, the objective of this study was to evaluate the cardioprotective effects of Krebs-Henseleit buffer (KHB), Bretschneider-HTK (HTK), St. Thomas No. 1 (STH-1), and Celsior (CEL) solutions infused at 10°C and 20°C. METHODS: Hearts isolated from male albino Wistar rats and prepared according to Langendorff were randomly divided equally into 8 groups according to the temperature of infusion (10°C or 20°C) and cardioprotective solutions (KHB, HTK, STH-1, and CEL). After stabilization with KHB at 37°C, baseline values were collected (control) for heart rate (HR), left ventricle systolic pressure (LVSP), coronary flow (CF), maximum rate of rise of left ventricular pressure during ventricular contraction (+dP/dt) and maximum rate of fall of left ventricular pressure during left ventricular relaxation (-dP/dt). The hearts were then perfused with cardioprotective solutions for 5 minutes and kept for 2 hours in static ischemia at 20°C. Data evaluation used analysis of variance (ANOVA) in all together randomized 2-way ANOVA and Tukey's test for multiple comparisons. The level of significance chosen was P < .05. RESULTS: We observed that all 4 solutions were able to recover HR, independent of temperature. Interestingly, STH-1 solution at 20°C showed HR above baseline throughout the experiment. An evaluation of the corresponding hemodynamic values (LVSP, +dP/dt, and -dP/dt) indicated that treatment with CEL solution was superior at both temperatures compared with the other solutions, and had better performance at 20°C. When analyzing performance on CF maintenance, we observed that it was temperature dependent. However, when applying both HTK and CEL, at 10°C and 20°C respectively, indicated better protection against development of tissue edema. Multiple comparisons between treatments and hemodynamic variable outcomes showed that using CEL solution resulted in significant improvement compared with the other solutions at both temperatures. CONCLUSION: The solutions investigated were not able to fully suppress the deleterious effects of ischemia and reperfusion of the heart. However, these results allow us to conclude that temperature and the cardioprotective solution are interdependent as far as myocardial protection. Although CEL solution is the best for in myocardial protection, more studies are needed to understand the interaction between temperature and perfusion solution used. This will lead to development of better and more efficient cardioprotective methods.

Echocardiographic and Doppler assessment of maternal cardiovascular function in normal and abnormal canine pregnancies.

The objective was to verify maternal hemodynamic differences between normal and abnormal pregnancies in dogs. Brucella-negative pregnant bitches (n = 31) were retrospectively classified into abnormal (which had either their pregnancy interrupted between Days 52 and 60 or perinatal death of more than 50% of the litter; n = 14) and normal (which had delivered healthy puppies at term; n = 17). These dogs were evaluated with echocardiography every 10 days from Days 0 to 60 of gestation (Day 0 = estimated day of LH peak). Systolic blood pressure was also assessed. At Day 50 of gestation, left ventricular free wall in systole increased in the normal but not in the abnormal group (P < 0.01). In contrast, end systolic stress (P < 0.01) and systolic blood pressure (P < 0.01) diminished only in normal animals. We concluded that signs of altered maternal cardiovascular adaptation to pregnancy may be predictors of obstetrical complications in dogs.

Electrocardiographic changes in normal and abnormal canine pregnancy.

The aim of this study was to describe the canine electrocardiographic changes in the course of normal and abnormal pregnancy. Twenty-three Brucellosis-negative pregnant bitches were retrospectively classified as normal (n = 12) or abnormal (n = 11). A control group of non-pregnant dioestrous bitches (n = 10) was also included. Normal pregnant females delivered healthy puppies at term while abnormal animals interrupted their pregnancy between days 52-60 (from estimated luteinizing hormone peak) or presented perinatal litter death higher than 60%. All the bitches were electrocardiographically evaluated every 10 days from day 0 to day 65 of the oestrous cycle, to parturition or abortion. Percentage heart rate change increased 31.3% from day 40 to 60 in normal gestation while it decreased -1.8% in dioestrous bitches, although it did not change in the abnormal group (p < 0.01). In the abnormal pregnant group but not in the others, percentage QRSa change fell to -34% on day 60 (p < 0.01). At the same time point, percentage QRSd change was 6.2% vs -4.9% in normal gestations and dioestrous animals, respectively (p < 0.05). Corrected QT interval augmented from day 40 onwards up to 9.9% and 4.3% in the normal pregnant and dioestrous groups, respectively, while it remained unchanged in abnormal gestations (p < 0.05). It is concluded that during normal canine pregnancy, some electrocardiographic parameters begin changing from day 40 onwards, and that pathological gestations differ from normality from day 30. The use of electrocardiography in canine obstetrics might contribute to identify abnormal outcomes before they become clinically evident.

Hypertensive effects of the iv administration of picomoles of ouabain.

Ouabain, an endogenous digitalis compound, has been detected in nanomolar concentrations in the plasma of several mammals and is associated with the development of hypertension. In addition, plasma ouabain is increased in several hypertension models, and the acute or chronic administration of ouabain increases blood pressure in rodents. These results suggest a possible association between ouabain and the genesis or development and maintenance of arterial hypertension. One explanation for this association is that ouabain binds to the α-subunit of the Na(+) pump, inhibiting its activity. Inhibition of this pump increases intracellular Na(+), which reduces the activity of the sarcolemmal Na(+)/Ca(2+) exchanger and thereby reduces Ca(2+) extrusion. Consequently, intracellular Ca(2+) increases and is taken up by the sarcoplasmic reticulum, which, upon activation, releases more calcium and increases the vascular smooth muscle tone. In fact, acute treatment with ouabain enhances the vascular reactivity to vasopressor agents, increases the release of norepinephrine from the perivascular adrenergic nerve endings and promotes increases in the activity of endothelial angiotensin-converting enzyme and the local synthesis of angiotensin II in the tail vascular bed. Additionally, the hypertension induced by ouabain has been associated with central mechanisms that increase sympathetic tone, subsequent to the activation of the cerebral renin-angiotensin system. Thus, the association with peripheral mechanisms and central mechanisms, mainly involving the renin-angiotensin system, may contribute to the acute effects of ouabain-induced elevation of arterial blood pressure.

Hypertensive effects of the iv administration of picomoles of ouabain

Ouabain, an endogenous digitalis compound, has been detected in nanomolar concentrations in the plasma of several mammals and is associated with the development of hypertension. In addition, plasma ouabain is increased in several hypertension models, and the acute or chronic administration of ouabain increases blood pressure in rodents. These results suggest a possible association between ouabain and the genesis or development and maintenance of arterial hypertension. One explanation for this association is that ouabain binds to the α-subunit of the Na+ pump, inhibiting its activity. Inhibition of this pump increases intracellular Na+, which reduces the activity of the sarcolemmal Na+/Ca2+ exchanger and thereby reduces Ca2+ extrusion. Consequently, intracellular Ca2+ increases and is taken up by the sarcoplasmic reticulum, which, upon activation, releases more calcium and increases the vascular smooth muscle tone. In fact, acute treatment with ouabain enhances the vascular reactivity to vasopressor agents, increases the release of norepinephrine from the perivascular adrenergic nerve endings and promotes increases in the activity of endothelial angiotensin-converting enzyme and the local synthesis of angiotensin II in the tail vascular bed. Additionally, the hypertension induced by ouabain has been associated with central mechanisms that increase sympathetic tone, subsequent to the activation of the cerebral renin-angiotensin system. Thus, the association with peripheral mechanisms and central mechanisms, mainly involving the renin-angiotensin system, may contribute to the acute effects of ouabain-induced elevation of arterial blood pressure.