Maria Visitación Bartolomé

Leptin induces cardiac fibrosis through galectin-3, mtor and oxidative stress: potential role in obesity

Objective: Leptin acts as a cardiac profibrotic factor. However, the mechanisms underlying this effect are unclear. Therefore, we sought to elucidate the mediators involved in this process and the potential role of leptin in cardiac fibrosis associated with obesity. Methods: Male Wistar rats were fed either a high-fat diet (HFD; 33.5% fat), or a standard diet (3.5% fat) for 6 weeks. Results: HFD animals show cardiac hypertrophy, fibrosis and an increase in O2- production as evaluated by dihydroethidium. Echocardiographic parameters of cardiac structure and systolic function were similar in both groups. Cardiac levels of leptin, collagen I, galectin-3 and transforming growth factor &bgr; (TGF-&bgr;) were higher in HFD than in controls. In cardiac myofibroblasts, leptin (10–100 ng/ml) increased O2−, collagen I, galectin-3, TGF-&bgr; and connective tissue growth factor production (CTGF). These effects were prevented by the presence of either melatonin (10−3 mmol/l) or the inhibitor of mTOR, rapamycin (10−4 mmol/l). Blockage of galectin-3 activity by N-acetyllactosamine (LacNac 10−3 mmol/l) reduced both collagen I and O2.− production induced by leptin. The p70S6 kinase activation/phosphorylation, the downstream mediator of mTOR, induced by leptin was not modified by melatonin. Leptin reduced the metalloproteinase (MMP) 2 activity and the presence of melatonin, rapamycin or LacNac were unable to prevent it. Conclusion: The data suggest that leptin locally produced in the heart could participate in the fibrosis observed in HFD by affecting collagen turnover. Collagen synthesis induced by leptin seems to be mediated by the production of galectin-3, TGF-&bgr; and CTGF through oxidative stress increased by activation of mTOR pathway.

Serotonergic innervation of the organ of corti of the cat cochlea

THE presence and distribution of serotonin-containing fibers within the adult cat cochlea is reported here for the first time. The 5-HT-like immunoreactive fibers were mainly found in the middle coils of the cat cochlea, following a peripheral distribution similar to that of the olivocochlear lateral efferent system (OLES). Thus, 5-HT-like immunoreactive varicose fibers were found within the intraganglionic spiral bundle, the inner and the tunnel bundles, although, some variation with the distribution of other neuroactive substances of the OLES were observed. In particular, some isolated fibers from the tunnel bundle branched towards the first row of the outer hair cells. Cochlear serotonergic fibers might be involved in the auditory processing within the cochlea as efferent modulating fibers.

Exposure to low mercury concentration in vivo impairs myocardial contractile function

Increased cardiovascular risk after mercury exposure has been described but cardiac effects resulting from controlled chronic treatment are not yet well explored. We analyzed the effects of chronic exposure to low mercury concentrations on hemodynamic and ventricular function of isolated hearts. Wistar rats were treated with HgCl₂ (1st dose 4.6 μg/kg, subsequent dose 0.07 μg/kg/day, im, 30 days) or vehicle. Mercury treatment did not affect blood pressure (BP) nor produced cardiac hypertrophy or changes of myocyte morphometry and collagen content. This treatment: 1) in vivo increased left ventricle end diastolic pressure (LVEDP) without changing left ventricular systolic pressure (LVSP) and heart rate; 2) in isolated hearts reduced LV isovolumic systolic pressure and time derivatives, and β-adrenergic response; 3) increased myosin ATPase activity; 4) reduced Na+-K+ ATPase (NKA) activity; 5) reduced protein expression of SERCA and phosphorylated phospholamban on serine 16 while phospholamban expression increased; as a consequence SERCA/phospholamban ratio reduced; 6) reduced sodium/calcium exchanger (NCX) protein expression and α-1 isoform of NKA, whereas α-2 isoform of NKA did not change. Chronic exposure for 30 days to low concentrations of mercury does not change BP, heart rate or LVSP but produces small but significant increase of LVEDP. However, in isolated hearts mercury treatment promoted contractility dysfunction as a result of the decreased NKA activity, reduction of NCX and SERCA and increased PLB protein expression. These findings offer further evidence that mercury chronic exposure, even at small concentrations, is an environmental risk factor affecting heart function.

The lysyl oxidase inhibitor (β-aminopropionitrile) reduces leptin profibrotic effects and ameliorates cardiovascular remodeling in diet-induced obesity in rats

Lysyl oxidase (LOX) is an extracellular matrix (ECM)-modifying enzyme that has been involved in cardiovascular remodeling. We explore the impact of LOX inhibition in ECM alterations induced by obesity in the cardiovascular system. LOX is overexpressed in the heart and aorta from rats fed a high-fat diet (HFD). β-Aminopropionitrile (BAPN), an inhibitor of LOX activity, significantly attenuated the increase in body weight and cardiac hypertrophy observed in HFD rats. No significant differences were found in cardiac function or blood pressure among any group. However, HFD rats showed cardiac and vascular fibrosis and enhanced levels of superoxide anion (O2(-)), collagen I and transforming growth factor β (TGF-β) in heart and aorta and connective tissue growth factor (CTGF) in aorta, effects that were attenuated by LOX inhibition. Interestingly, BAPN also prevented the increase in circulating leptin levels detected in HFD fed animals. Leptin increased protein levels of collagen I, TGF-β and CTGF, Akt phosphorylation and O2(-) production in both cardiac myofibroblasts and vascular smooth muscle cells in culture, while LOX inhibition ameliorated these alterations. LOX knockdown also attenuated leptin-induced collagen I production in cardiovascular cells. Our findings indicate that LOX inhibition attenuates the fibrosis and the oxidative stress induced by a HFD on the cardiovascular system. The reduction of leptin levels by BAPN in vivo and the ability of this compound to inhibit leptin-induced profibrotic mediators and ROS production in cardiac and vascular cells suggest that interactions between leptin and LOX regulate downstream events responsible for myocardial and vascular fibrosis in obesity.

DIOL Triterpenes Block Profibrotic Effects of Angiotensin II and Protect from Cardiac Hypertrophy

Background The natural triterpenes, erythrodiol and uvaol, exert anti-inflammatory, vasorelaxing and anti-proliferative effects. Angiotensin II is a well-known profibrotic and proliferative agent that participates in the cardiac remodeling associated with different pathological situations through the stimulation and proliferation of cardiac fibroblasts. Therefore, the aim of the study was to investigate the preventive effects of the natural triterpenes erythrodiol and uvaol on the proliferation and collagen production induced by angiotensin II in cardiac myofibroblasts. Their actions on cardiac hypertrophy triggered by angiotensin II were also studied. Methodology/Principal Findings The effect of erythrodiol and uvaol on angiotensin II-induced proliferation was evaluated in cardiac myofibroblasts from adult rats in the presence or the absence of the inhibitors of PPAR-γ, GW9662 or JNK, SP600125. The effect on collagen levels induced by angiotensin II was evaluated in cardiac myofibroblasts and mouse heart. The presence of low doses of both triterpenes reduced the proliferation of cardiac myofibroblasts induced by angiotensin II. Pretreatment with GW9662 reversed the effect elicited by both triterpenes while SP600125 did not modify it. Both triterpenes at high doses produced an increase in annexing-V binding in the presence or absence of angiotensin II, which was reduced by either SP600125 or GW9662. Erythrodiol and uvaol decreased collagen I and galectin 3 levels induced by angiotensin II in cardiac myofribroblasts. Finally, cardiac hypertrophy, ventricular remodeling, fibrosis, and increases in myocyte area and brain natriuretic peptide levels observed in angiotensin II-infused mice were reduced in triterpene-treated animals. Conclusions/Significance Erythrodiol and uvaol reduce cardiac hypertrophy and left ventricle remodeling induced by angiotensin II in mice by diminishing fibrosis and myocyte area. They also modulate growth and survival of cardiac myofibroblasts. They inhibit the angiotensin II-induced proliferation in a PPAR-γ-dependent manner, while at high doses they activate pathways of programmed cell death that are dependent on JNK and PPAR-γ.

HPLC detection of serotonin within the rat cochlea.

THIS study was performed to analyse the cochlear concentrations of serotonin (5-HT) and 5-hydroxyin dole-3-acetic acid (5-HIAA), their sources and modifications induced by noise exposure. Superior cervical ganglionectomy did not modify these concentrations. However, removal of the blood by aortic perfusion reduced significantly (about 76%) the cochlear concentration of 5-HT without affecting the 5-HIAA concentration. These results indicate that blood constitutes an important source of 5-HT to the cochlea, opposite to the superior cervical ganglion. Exposure to noise at 90 dB SPL did not modify the total cochlear concentrations of 5-HT and 5-HIAA, or the concentrations remaining after removal of the blood, suggesting that 5-HT could have a modulatory role in the cochlea distinct from that of olivocochlear neurotransmitters.

Decalcification by ascorbic acid for immuno- and affinohistochemical techniques on the inner ear.

Abstract An ascorbic acid decalcifying solution was applied to immuno- and affinohistochemical studies on the inner ear. Rat inner ears fixed in 4% paraformaldehyde in PBS or in 2% acetic acid in ethanol solutions were adequately decalcified in an ascorbic acid solution, at a temperature of 4°C. The decalcifying solution was prepared with 1% ascorbic acid and 0.84% sodium chloride in distilled water (pH 2.5–2.6). The decalcification time was in a direct relationship to the specimen calcification. In this study, two neuroactive substances (γ-aminobutyric acid and calcitonin gene-related peptide), neurofilaments, and the galectine endogenous lectin were successfully detected immunohistochemically.

Cellular and molecular bases of neuroplasticity: brainstem effects after cochlear damage

After a cochlear lesion or auditory nerve damage, afferent connections from auditory ganglia can be highly altered. This results in a clear reduction of auditory input and an alteration of connectivity of terminals on cochlear nuclei neurons. Such a process could stimulate the reorganization of the neural circuits and neuroplasticity. Cochlea removal has been demonstrated to be a good model in which to analyse brainstem neuroplasticity, particularly with regard to the cochlear nuclei. After cochlea removal three main periods of degeneration and regeneration were observed. Early effects, during the first week post lesion, involved acute degeneration with nerve ending oedema and degeneration. During the second and, probably, the third post lesion weeks, degeneration was still present, even though a limited and diffuse expression of GAP-43 started. Around 1 month post lesion, degeneration at the cochlear nuclei progressively disappeared and a relevant GAP-43 expression was found. We conclude that neuroplasticity leads neurons to modify their activity and/or their synaptic tree as a consequence of animal adaptation to learning and memory. For the human being neuroplasticity is involved in language learning and comprehension, particularly the acquisition of a second language. Neuroplasticity is important for therapeutic strategies, such as hearing aids and cochlear implants.

Immunocytochemical detection of synaptophysin in C57BL/6 mice cochlea during aging process

Aged mammals frequently exhibit a bilateral, progressive, and symmetric deafness related to the degeneration of auditory receptor. However, little is still known about aging effects on synapses in this receptor. Synaptophysin (Syp) is a 38 kDa Ca2+ binding glycoprotein widely found in presynaptic membrane and vesicles. The Syp has been found in presynaptic buttons of efferent auditory fibers, within the developing and adult auditory receptor. The detection of Syp in aged cochleae could provide relevant information about synaptic changes and receptor degeneration process observed in old animals. This paper focuses on aging linked changes related to the presence of Syp in cochleae of C57BL/6J mice (from 1 to 24 months old). Results showed that during the first months of age, no significant changes were observed in the Syp distribution under the basal pole of inner (IHCs) neither the outer (OHCs) hair cells. At six months of age, a significant decrease of Syp immunocytochemical detection appeared in fibers under the most external row of OHCs, but restricted to the cochlear basal coil. Only a very scarce reduction of Syp was noted under the IHC and the other OHC rows, also at the basal coil. From mice 9 months old on, a progressive decrease of the presence of Syp was found under IHC and all OHC rows starting at the basal coil and reaching the apical coil in the oldest mice. All these data could indicate that the cochlea aging process early affects to presynaptic membrane proteins of efferent endings fibers. This early alteration of cochleae efferent synapses could be involved in the whole degeneration of the Cortis organ.

Galectine-1 expression in cochleae of C57BL/6 mice during aging.

Presbycusis is a progressive hearing loss related to the aging process associated with auditory receptor degeneration. Adult animals exhibited galectine-1 (GAL-1) expression in epithelial cells of the inner and outer spiral sulci and Hensen cells, which was absent from the sensory and supporting cells. The progressive age-related degeneration of auditory receptor was analyzed using an anti-GAL-1 monoclonal antibody on cochleae of C57BL/6 mice (1–24 months old). Mice ⩽ 9 months old showed adult and healthy auditory receptors, with a similar GAL-1 expression along the time. Conversely, animals > 1 year old exhibited a sensory degeneration at the cochleae basal coil that progressively reached the middle coil. Older animals (18–24 months old) showed a single layer epithelium that replaced the auditory receptor. The age-related degenerative process of the auditory receptor involves sensory but also supporting cells, which are both substituted by epithelial non-specialized cells.