Carmen Lopez-Sanchez

Complex I and cytochrome c are molecular targets of flavonoids that inhibit hydrogen peroxide production by mitochondria.

Flavonoids can protect cells from different insults that lead to mitochondria-mediated cell death, and epidemiological data show that some of these compounds attenuate the progression of diseases associated with oxidative stress and mitochondrial dysfunction. In this work, a screening of 5 flavonoids representing major subclasses showed that they display different effects on H₂O₂ production by mitochondria isolated from rat brain and heart. Quercetin, kaempferol and epicatechin are potent inhibitors of H₂O₂ production by mitochondria from both tissues (IC₅₀ approximately 1-2 μM), even when H₂O₂ production rate was stimulated by the mitochondrial inhibitors rotenone and antimycin A. Although the rate of oxygen consumption was unaffected by concentrations up to 10 μM of these flavonoids, quercetin, kaempferol and apigenin inhibited complex I activity, while up to 100 μM epicatechin produced less than 20% inhibition. The extent of this inhibition was found to be dependent on the concentration of coenzyme Q in the medium, suggesting competition between the flavonoids and ubiquinone for close binding sites in the complex. In contrast, these flavonoids did not significantly inhibit the activity of complexes II and III, and did not affect the redox state of complex IV. However, we have found that epicatechin, quercetin and kaempferol are able to stoichiometrically reduce purified cytochrome c. Our results reveal that mitochondria are a plausible main target of flavonoids mediating, at least in part, their reported preventive actions against oxidative stress and mitochondrial dysfunction-associated pathologies.

Developmental regulation of a proinsulin messenger RNA generated by intron retention

Proinsulin gene expression regulation and function during early embryonic development differ remarkably from those found in postnatal organisms. The embryonic proinsulin protein content decreased from gastrulation to neurulation in contrast with the overall proinsulin messenger RNA increase. This is due to increasing levels of a proinsulin mRNA variant generated by intron 1 retention in the 5′ untranslated region. Inclusion of intron 1 inhibited proinsulin translation almost completely without affecting nuclear export or cytoplasmic decay. The novel proinsulin mRNA isoform expression was developmentally regulated and tissue specific. The proportion of intron retention increased from gastrulation to organogenesis, was highest in the heart tube and presomitic region, and could not be detected in the pancreas. Notably, proinsulin addition induced cardiac marker gene expression in the early embryonic stages when the translationally active transcript was expressed. We propose that regulated unproductive splicing and translation is a mechanism that regulates proinsulin expression in accordance with specific requirements in developing vertebrates.

Localization of Cells of the Prospective Neural Plate, Heart and Somites within the Primitive Streak and Epiblast of Avian Embryos at Intermediate Primitive-Streak Stages

By constructing avian transplantation chimeras using fluorescently-labeled grafts and antibodies specific for grafted cells, we have generated a prospective fate map of the primitive streak and epiblast of the avian blastoderm at intermediate primitive-streak stages (stages 3a/3b). This high-resolution map confirms our previous study on the origin of the cardiovascular system from the primitive streak at these stages and provides new information on the epiblast origin of the neural plate, heart and somites. In addition, the origin of the rostral endoderm is now documented in more detail. The map shows that the prospective neural plate arises from the epiblast in close association with the rostral end of the primitive streak and lies within an area extending 250 µm rostral to the streak, 250 µm lateral to the streak and 125 µm caudal to the rostral border of the streak. The future floor plate of the neural tube arises within the midline just rostral to the streak, confirming our earlier study, but unlike at the late-primitive streak stages when both Hensen’s node and the midline area rostral to Hensen’s node contribute to the floor plate, only the area rostral to the primitive streak contributes to the floor plate at intermediate primitive-streak stages. Instead of contributing to the floor plate of the neural tube, the rostral end of the primitive streak at intermediate primitive-streak stages forms the notochord as well as the rostromedial endoderm, which lies beneath the prechordal plate mesoderm and extends caudolaterally on each side toward the cardiogenic areas. The epiblast lateral to the primitive streak and caudal to the neural plate contributes to the heart and it does so in rostrocaudal sequence (i.e., rostral grafts contribute to rostral levels of the straight heart tube, whereas progressively more caudal grafts contribute to progressively more caudal levels of the straight heart tube), and individual epiblast grafts contribute cells to both the myocardium and endocardium. The prospective somites (i.e., paraxial mesoderm) lie within the epiblast just lateral to the prospective heart mesoderm. Comparing this map with that constructed at late primitive-streak stages reveals that by the late primitive-streak stages, prospective heart mesoderm has moved from the epiblast through the primitive streak and into the mesodermal mantle, and that some of the prospective somitic mesoderm has entered the primitive streak and is undergoing ingression.

Kaempferol protects against rat striatal degeneration induced by 3‐nitropropionic acid

3‐Nitropropionic acid (NPA) produces degeneration of striatum and some neurological disturbances characteristic of Huntington’s disease in rodents and primates. We have shown that the flavonoid kaempferol largely reduced striatal damage induced by cerebral ischaemia‐reperfusion in rats ( Lopez‐Sanchez et al. 2007 ). In this work, we report that intraperitoneal (i.p.) administration of kaempferol affords an efficient protection against NPA‐induced neurodegeneration in Wistar rats. We studied the effects of daily i.p. injections of 7, 14 and 21 mg of kaempferol/kg body weight during the NPA‐treatment (25 mg/kg body weight/12 h i.p., for 5 days) on the neurological deficits, degeneration of rat striatum and oxidative stress markers. Intraperitoneal injections of 14–21 mg of kaempferol/kg body weight largely attenuated motor deficit and delayed mortality. The higher dose of kaempferol prevented the appearance of NPA‐induced striatal lesions up to the end of treatment, as revealed by haematoxylin‐eosin and TUNEL staining, and also NPA‐induced oxidative stress, because it blocked the fall of reduced glutathione and the increase of protein nitrotyrosines in NPA‐treated rats. It was found that striatal degeneration was associated with calpains activation and a large inactivation of creatine kinase, which were also prevented when the higher doses of kaempferol were administered.

Induction of cardiogenesis by Hensen's node and fibroblast growth factors

Abstract. The earliest events underlying cardiac induction and morphogenesis remain largely unknown. In the present study, we show that Hensens node, the organizer of the avian embryo, induces cardiogenesis. Specifically, following heterotopic transplantation, Hensens node induces ectopic host tissue that expresses two early cardiac markers (cNkx-2.5 and cNkx-2.8), as well as a ventricular marker (VMHC1), but not an atrial marker (AMHC1). Moreover, we examine the potential roles of candidate growth factors known to be secreted by Hensens node. Our results show that fibroblast growth factors (FGF-2 and FGF-4) when ectopically expressed can initiate cardiac development, inducing host tissue to express the two cardiac transcription factors cNkx-2.5 and cNkx-2.8, as well as the cardiac-restricted structural gene VMHC1, but not AMHC1. In contrast to FGFs, TGFβ family members fail to induce ectopic tissue and expression of cardiac marker genes. We also examined the effects of growth factors on the morphogenesis of the host embryos heart. Both exogenous FGFs and TGFβ family members perturb normal morphogenesis of the early cardiac tube and alter patterns of ventricular and atria gene expression in characteristic ways. Namely, exogenous FGFs expand areas expressing the ventricular marker VMHC1 at the expense of areas expressing the atrial marker AMHC1. Conversely, exogenous TGFβ1 inhibits expression of VMHC1, expanding AMHC1 expression. We show here that Hensens node and FGFs induce ectopic expression of cardiac lineage markers, and that FGF and TGFβ family members can modulate early development of the heart. Collectively, these data suggest that the organizer plays a crucial role in cardiac induction and morphogenesis, mediated in part by endogenous members of the FGF and TGFβ families.

Transarterial Prostatic Embolization: Initial Experience in a Canine Model

OBJECTIVE The purpose of this study was to prospectively evaluate pathologic responses to transarterial prostatic embolization and its technical safety in a canine model. MATERIALS AND METHODS Ten adult male beagle dogs were surgically castrated and given hormonal therapy for 4 months to induce prostatic hyperplasia. After three months of hormonal therapy, the dogs were randomly assigned to a transarterial prostatic embolization group (n = 7) or a control group (n = 3). Dogs in the transarterial prostatic embolization group were subjected to embolization with microspheres 300-500 μm in diameter. Four months after the study was begun, all dogs were sacrificed for pathologic study. Transrectal ultrasound and MRI were performed to evaluate pathologic responses. The data on prostate size acquired with transrectal ultrasound were processed for statistical analysis by paired Student t test. RESULTS The canine prostatic hyperplasia model was successfully established in 10 dogs. The increase in mean prostate size being as great as 572% after 3 months of hormonal therapy. An intraprostatic cavity was detected 1 month after transarterial prostatic embolization in all seven dogs. Four dogs had significant shrinkage of the prostate, and the other three had an increase in prostate size. Imaging examinations and necropsy revealed a huge cavity occupying almost the entire prostate in the three dogs with increased prostate size. No complications associated with transarterial prostatic embolization were encountered. CONCLUSION Transarterial prostatic embolization is a safe procedure that can induce prostatic infarction and ablate the prostate. The findings suggest the procedure has potential clinical applications in the care of patients with benign prostatic hyperplasia.

Rapid triple‐labeling method combining in situ hybridization and double immunocytochemistry

A new, rapid method is described for combining in situ hybridization and immunocytochemistry to define cell populations and to map three‐dimensional movements of groups of labeled cells within developing chick embryos. The method allows fluorescently labeled cells to be followed in living embryos and subsequently detected as a permanent reaction product for detailed three‐dimensional analysis by immunocytochemistry in histological serial sections. Cell identity can be ascertained using a specific riboprobe and in situ hybridization. With this approach, the movements of two groups of cells can be mapped simultaneously (using two different fluorescent trackers and, subsequently, two different chromogens for immunocytochemistry) to analyze relative movements within an embryo, and when combined with in situ hybridization with a specific riboprobe for cell identity, allows fate mapping studies to be conducted using molecular criteria, rather than solely at morphological/positional criteria. The improved method enables the investigator to extract substantially more information from individual embryos, maximizing the results obtained from labor‐intensive fate mapping studies. Developmental Dynamics 230:309–315, 2004.

Movement and commitment of primitive streak precardiac cells during cardiogenesis

Fate maps are required to address questions about the commitment and differentiation of precardiac cells. Here, we report a detailed study of the precardiac cells located at the level of the primitive streak, employing different experiments with a variety of techniques combining double transplantations, microinjections and immunocytochemistry. Most cells of the more rostral segments of the primitive streak were found to contribute cells to the endodermal layer, adjacent to precardiac mesodermal cells of the heart forming region whose provenance was in the immediately more caudal segments of the primitive streak. We established a close spatio-temporal relationship between the two cell layers and the expression of their specific cardiac markers (cNkx-2.5, Bmp2, Cripto, Usmaar, dHand, GATA4, Pitx2, Hex, Fgf8, AMHC1 and VMHC1). We also analyzed the ability of precardiac cells to differentiate when they are transplanted to ectopic locations or are subjected to the influence of the organizer. We propose that the precardiac cells of the primitive streak form at least two groups with different significance. One, regulated by mediation of the organizer, is located preferentially in the more rostral region of the primitive streak. It consists of the prospective cells of the endoderm layer, with a hierarchic pattern of expression of different genes characterized by its capacity for induction and regulation of a second group of cells. This second group is located preferentially in the more caudal segments, and is fated to form the precardiac mesoderm, whose differentiation would be characterized by the expression of various specific genes.

MiR-23b and miR-199a impair epithelial-to-mesenchymal transition during atrioventricular endocardial cushion formation

Background: Valve development is a multistep process involving the activation of the cardiac endothelium, epithelial‐mesenchymal transition (EMT) and the progressive alignment and differentiation of distinct mesenchymal cell types. Several pathways such as Notch/delta, Tgf‐beta and/or Vegf signaling have been implicated in crucial steps of valvulogenesis. We have previously demonstrated discrete changes in microRNAs expression during cardiogenesis, which are predicted to target Bmp‐ and Tgf‐beta signaling. We now analyzed the expression profile of 20 candidate microRNAs in atrial, ventricular, and atrioventricular canal regions at four different developmental stages. Results: qRT‐PCR analyses of microRNAs demonstrated a highly dynamic and distinct expression profiles within the atrial, ventricular, and atrioventricular canal regions of the developing chick heart. miR‐23b, miR‐199a, and miR‐15a displayed increased expression during early AVC development whereas others such as miR‐130a and miR‐200a display decreased expression levels. Functional analyses of miR‐23b, miR‐199a, and miR‐15a overexpression led to in vitro EMT blockage. Molecular analyses demonstrate that distinct EMT signaling pathways are impaired after microRNA expression, including a large subset of EMT‐related genes that are predicted to be targeted by these microRNAs. Conclusions: Our data demonstrate that miR‐23b and miR‐199a over‐expression can impair atrioventricular EMT. Developmental Dynamics 244:1259–1275, 2015.

Tyrosine hydroxylase is expressed during early heart development and is required for cardiac chamber formation

AIMS Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in catecholamine biosynthesis. Whereas the neuroendocrine roles of cathecolamines postnatally are well known, the presence and function of TH in organogenesis is unclear. The aim of this study was to define the expression of TH during cardiac development and to unravel the role it may play in heart formation. METHODS AND RESULTS We studied TH expression in chick embryos by whole mount in situ hybridization and by quantitative reverse transcription-polymerase chain reaction and analysed TH activity by high-performance liquid chromatography. We used gain- and loss-of-function models to characterize the role of TH in early cardiogenesis. We found that TH expression was enriched in the cardiac field of gastrulating chick embryos. By stage 8, TH mRNA was restricted to the splanchnic mesoderm of both endocardial tubes and was subsequently expressed predominantly in the myocardial layer of the atrial segment. Overexpression of TH led to increased atrial myosin heavy chain (AMHC1) and T-box 5 gene (Tbx5) expression in the ventricular region and induced bradyarrhythmia. Similarly, addition of l-3,4-dihydroxyphenylalanine (l-DOPA) or dopamine induced ectopic expression of cardiac transcription factors (cNkx2.5, Tbx5) and AMHC1 as well as sarcomere formation. Conversely, blockage of dopamine biosynthesis and loss of TH activity decreased AMHC1 and Tbx5 expression, whereas exposure to retinoic acid (RA) induced TH expression in parallel to that of AMHC1 and Tbx5. Concordantly, inhibition of endogenous RA synthesis decreased TH expression as well as that of AMHC1 and Tbx5. CONCLUSION TH is expressed in a dynamic pattern during the primitive heart tube formation. TH induces cardiac differentiation in vivo and it is a key regulator of the heart patterning, conferring atriogenic identity.