Endocardial-mesenchymal transition underlies fusion of the conotruncal ridges during embryonic cardiac outflow tract septation

The embryonic cardiac outflow tract (conotruncus) is a single tubular chamber that connects the right ventricle with the aortic arch arteries. It contains two opposite, long and helical mesenchymal cushions covered by endocardial cells (conotruncal ridges). Conotruncal division (septation) gives rise to the adult right and left outflows together with the aortic and pulmonary valves. It takes place by fusion of the two opposite ridges and formation of the conotruncal septum. Although the participation of neural crest cells in septation is well established, the mechanism of fusion of the conotruncal ridges remains unknown. Defects in fusion have been shown to produce bicuspid aortic valve, the most prevalent human congenital cardiac malformation, in a hamster model. Three fusion mechanisms have been proposed to operate during embryonic development: epithelial adhesion, epithelial apoptosis and epithelial-mesenchymal transition (EMT). The first mechanism entails the expression of adhesion molecules and the maintenance of the identity of cells in contact, whereas in the other two, epithelial cells covering the fusing structures disappear by apoptosis or by transforming into mesenchymal cells. The objective of this study is to elucidate the mechanism involved in the fusion of the conotruncal ridges. Immunofluorecence techniques were used in ED 11-12 hamster embryos. The results indicate that the mechanism of EMT, but not epithelial adhesion or apoptosis, is involved in the process of fusion of the conotruncal ridges. The EMT mechanism associated with conotruncal septation seems to be uncoupled from the process of formation of the endocardial cushions, which takes place at early stages. With these results, we can raise the hypothesis that defects in the EMT process may lead to different morphological types of bicuspid aortic valve.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This study was supported by P10-CTS-6068 (Junta de Andalucía), CGL2014-52356-P and CGL2017-85090-P (Ministerio de Economía y Competitividad), contract UMAJI75 (Junta de Andalucía, European Social Fund), and Universidad de Málaga

Is the bulbus arteriosus of fish homologous to the mamalian intrapericardial thoracic arteries?

El resumen aparece en el Program & Abstracts of the 10th International Congress of Vertebrate Morphology, Barcelona 2013.Anatomical Record, Volume 296, Special Feature — 1: P-089.Two major findings have significantly improved our understanding of the embryology and evolution of the arterial pole of the vertebrate heart (APVH): 1) a new embryonic presumptive cardiac tissue, named second heart field (SHF), forms the myocardium of the outflow tract, and the walls of the ascending aorta (AA) and the pulmonary trunk (PT) in mammals and birds; 2) the bulbus arteriosus (BA), previously thought to be an actinopterygian apomorphy, is present in all basal Vertebrates, and probably derives from the SHF. We hypothesized that the intrapericardial portions of the AA and the PT of mammals are homologous to the BA of basal vertebrates. To test this, we performed 1) a literature review of the anatomy and embryology of the APVH; 2) novel anatomical, histomorphological, and embryological analyses of the APVH, comparing basal (Galeus atlanticus), with apical (Mus musculus and Mesocricetus auratus) vertrebrates. Evidence obtained: 1) Anatomically, BA, AA, and PT are muscular tubes into the pericardial cavity, which connect the distal myocardial outflow tracts with the aortic arch system. Coronary arteries run through or originate at these anatomical structures; 2) Histologically, BA, AA, and PT show an inner layer of endothelium covered by circumferentially oriented smooth muscle cells, collagen fibers, and lamellar elastin. The histomorphological differences between the BA and the ventral aorta parallel those between intrapericardial and extrapericardial great arteries; 3) Embryologically, BA, AA, and PT are composed of smooth muscle cells derived from the SHF. They show a similar mechanism of development: incorporation of SHF‐derived cells into the pericardial cavity, and distal‐to‐proximal differentiation into an elastogenic cell linage. In conclusion, anatomical, histological and embryological evidence supports the hypothesis that SHF is a developmental unit responsible for the formation of the APVH. The BA and the intrapericardial portions of the great arteries must be considered homologous structures.Proyecto P10-CTS-6068 (Junta de Andalucía); proyecto CGL-16417 (Ministerio de Ciencia e Innovación); Fondos FEDER

The anatomical components of the cardiac outflow tract of the bichir, polypterus senegalus. Evolutionary significance

El resumen aparece en el Program & Abstracts of the 10th International Congress of Vertebrate Morphology, Barcelona 2013.Anatomical Record, Volume 296, Special Feature — 1: P-077.In chondrichthyans and actinopterygians, the outflow tract of the heart, namely, the cardiac portion intercalated between the ventricle and the ventral aorta, consists of two anatomical components: conus arteriosus and bulbus arteriosus. In chondrichthyans and extant representatives of phylogenetically ancient actinopterygian groups, the conus and bulbus are well-developed in size, whereas in most teleosts, the bulbus is markedly larger than the conus. Current knowledge about the cardiac outflow tract of the polypteriformes is scarce and highly contradictory, a fact that contrasts with their crucial phylogenetic position at the source of the actinopterygian lineage. In fact, it remains uncertain whether they have a bulbus at the arterial pole of the heart. The present study aimed to elucidate the anatomical arrangement of the cardiac outflow tract of the bichir in an attempt to improve our understanding of the evolution of the vertebrate heart. We examined the hearts from 12 bichirs using histochemical and immunohistochemical techniques. Our findings showed that the outflow tract of the bichir consists of two components, namely, a long conus arteriosus, largely composed of myocardial tissue and furnished with a variable number of valves at its luminal side, and a very short, elastin rich bulbus arteriosus, devoid of myocardium. The bulbus has an arterial-like histological composition. However, it differs from the aorta because it has a thicker wall, shows a different arrangement of the histological elements, is covered by the epicardium and is crossed by coronary arteries. The present observations are consistent with the notion that the conus arteriosus and the bulbus arteriosus have coexisted from the beginning of the jawed vertebrate radiation. This is of particular interest, because there is evidence that the bulbus arteriosus, which is a second heart field derivative, is homologous with the intrapericardial portions of the aorta and pulmonary artery of birds and mammals.Proyecto CGL2010-16417/BOS; Fondos FEDER

Bicuspid Aortic Valves With Different Spatial Orientations of the Leaflets Are Distinct Etiological Entities

ObjectivesThe aim of this study was to decide whether bicuspid aortic valves (BAVs) with fused right and noncoronary leaflets (R-N) and BAVs with fused right and left leaflets (R-L) have different etiologies or are the product of a single diathesis.BackgroundThe BAV is the most common congenital cardiac malformation. The R-N and R-L BAVs are the most frequent BAV subtypes.MethodsThe study was carried out in adult and embryonic hearts of endothelium nitric oxide synthase knock-out mice and inbred Syrian hamsters with a high incidence of R-N and R-L BAVs, respectively. The techniques used were histochemistry, immunohistochemistry, and scanning electron microscopy.ResultsThe R-N BAVs result from a defective development of the cardiac outflow tract (OT) endocardial cushions that generates a morphologically anomalous right leaflet. The left leaflet develops normally. The R-L BAVs are the outcome of an extrafusion of the septal and parietal OT ridges that thereby engenders a sole anterior leaflet. The noncoronary leaflet forms normally.ConclusionsThe R-N and R-L BAVs are different etiological entities. The R-N BAVs are the product of a morphogenetic defect that happens before the OT septation and that probably relies on an exacerbated nitric oxide–dependent epithelial-to-mesenchymal transformation. The R-L BAVs result from the anomalous septation of the proximal portion of the OT, likely caused by a distorted behavior of neural crest cells. Care should be taken in further work on BAV genetics because R-N and R-L BAVs might rely on different genotypes. Detailed screening for R-N and R-L BAVs should be performed for a better understanding of the relationships between these BAV morphologic phenotypes and other heart disease

Coronary artery high take-off in rodents and the possible involvement of Smad2

The coronary arteries (CAs) supply the mammalian heart with oxygenated blood. They arise from the right and left aortic valve sinuses at the aortic root. In humans, the occurrence of a CA arising ectopically from the tubular aorta, a condition called high take-off (HTO), is rare (A (Smad2C>A) allele is associated with HTO in this species. In order to test whether HTO occurs in association with Smad2C>A in other rodents, we examined the anatomical origin of the CAs, by means of stereomicroscopy and a corrosion cast technique, in 3,388 specimens belonging to 17 rodent species. In addition, Smad2 DNA sequence from M. musculus was compared by Blastn analyses with that from six of the species examined in which this sequence is known (Mus spretus, Rattus norvegicus, Apodemus sylvaticus, Myodes glareolus, Mesocricetus auratus, Microtus agrestis). HTO occurred in nine out of 17 species studied. The incidence of HTO ranged from 0.4% to 6.5% (low) in three species and from 15.9% to 25% (high) in six species. The Smad2 sequence showed similarities higher than 75% for the whole gene, and higher than 71% for the intron sequence that includes the rs29725537:C>A Single Nucleotide Polymorphism. The Smad2C>A allele was identified only in M. spretus and A. sylvaticus, with high incidences of HTO, whereas R. norvegicus, M. glareolus, M. auratus and M. agrestis showed low or null incidences. We conclude that HTO is a common trait in rodents, which does not lead to cardiac pathology probably due to the intramyocardial condition of their CAs, as opposed to the human subepicardial CAs. The Smad2C>A allele may be involved in the development of HTO and probably other phenotypes in different rodent species.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Funding: CGL2017-85090-P, FPU15/03209, UMAJI75 and FEDER

Experimental evidence of the genetic hypothesis on the etiology of bicuspid aortic valve aortopathy in the hamster model.

Bicuspid aortopathy occurs in approximately 50% of patients with bicuspid aortic valve (BAV), the most prevalent congenital cardiac malformation. Although different molecular players and etiological factors (genetic and hemodynamic) have been suggested to be involved in aortopathy predisposition and progression, clear etiophysiopathological mechanisms of disease are still missing. The isogenic (genetically uniform) hamster (T) strain shows 40% incidence of BAV, but aortic dilatations have not been detected in this model. We have performed comparative anatomical, histological and molecular analyses of the ascending aorta of animals with tricuspid aortic valve (TAV) and BAV from the T strain (TTAV and TBAV, respectively) and with TAV from a control strain (HTAV). Aortic diameter, smooth muscle apoptosis, elastic waviness, and Tgf-β and Fbn-2 expression were significantly increased in T strain animals, regardless of the valve morphology. Strain and aortic valve morphology did not affect Mmp-9 expression, whereas Mmp-2 transcripts were reduced in BAV animals. eNOS protein amount decreased in both TBAV and TTAV compared to HTAV animals. Thus, histomorphological and molecular alterations of the ascending aorta appear in a genetically uniform spontaneous hamster model irrespective of the aortic valve morphology. This is a direct experimental evidence supporting the genetic association between BAV and aortic dilatation. This model may represent a population of patients with predisposition to BAV aortopathy, in which increased expression of Tgf-β and Fbn-2 alters elastic lamellae structure and induces cell apoptosis mediated by eNOS. Patients either with TAV or BAV with the same genetic defect may show the same risk to develop bicuspid aortopathy.This work was supported by Consejería de Salud y Familias, Junta de Andalucía (PI-0530-2019), Consejería de Economía y Conocimiento, Junta de Andalucía (UMA20-FEDERJA-041), Ministerio de Ciencia e Innovación (grants CGL2017-85090- P and PT20/00101, fellowship PRE2018-083176 to MS-N), and FEDER funds.S

El origen anatómico de la arteria coronaria septal del hámster sirio es un carácter heredable

Este artículo se ha publicado en: Rev Esp Cardiol 2016, 69, supl. 1:683.Introducción y objetivos: En los roedores, incluidos los empleados en el laboratorio, las arterias coronarias son intramiocárdicas. Normalmente se distinguen 3 arterias coronarias, la derecha, que irriga el corazón derecho, la izquierda, que irriga el corazón izquierdo, y la septal, que irriga el septo interventricular. En el hámster sirio, una especie empleada como modelo en estudios sobre anomalías congénitas de las arterias coronarias, la arteria septal se origina de la coronaria derecha o de la izquierda, o de un ostium propio en el seno aórtico correspondiente. El objetivo fue averiguar si el origen anatómico de la arteria septal del hámster está sujeto a factores hereditarios. Métodos: Se estudiaron moldes internos del corazón de un total de 483 hámsteres, seleccionados de 74 camadas y sus progenitores. Todos los animales tenían arterias coronarias normales. Con los datos obtenidos, se calculó la incidencia del origen de la arteria septal (izquierda o derecha) en individuos cuyos parentales presentaban (1) ambos septal izquierda, (2) ambos septal derecha y (3) uno septal derecha y el otro izquierda. Mediante el test de χ2, se verificó la hipótesis de que el origen de la arteria septal de un individuo es independiente del que presentan sus parentales. Resultados: La proporción de arterias septales derecha e izquierda varió significativamente dependiendo del fenotipo parental (χ2 = 33,121, p < 0,001). La arteria septal izquierda fue más frecuente de lo esperado en animales cuyos parentales tenían ambos arteria septal izquierda (p < 0,01), mientras que la arteria septal derecha lo era en animales en los que uno (p < 0,001) o ambos (p < 0,001) parentales tenían arteria septal derecha. Conclusiones: Los presentes resultados son los primeros en demostrar la influencia de factores genéticos en las variaciones anatómicas de un patrón arterial coronario, en particular de la arteria septal del hámster. Estos hallazgos son consistentes con el hecho de que, en humanos, determinadas características morfológicas del árbol arterial coronario asociadas a la enfermedad coronaria tienen un componente genético patente. El estudio del sustrato genético que subyace al establecimiento de los patrones anatómicos de las arterias coronarias en roedores puede contribuir a identificar marcadores de susceptibilidad a la enfermedad coronaria humana.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PI-0888-2012 (Junta de Andalucía). Red de Investigación Cardiovascular (RIC; RETICs

The Medical versus Zoological Concept of Outflow Tract Valves of the Vertebrate Heart.

The anatomical elements that in humans prevent blood backflow from the aorta and pulmonary artery to the left and right ventriclesare the aortic and pulmonary valves, respectively. Each valve regularly consists of three leaflets (cusps), each supported by its valvular sinus. From the medical viewpoint, each set of three leaflets and sinuses is regarded as a morpho-functional unit. This notion also applies to birds and non-human mammals. However, the structures that prevent the return of blood to the heart in other vertebrates are notably different. This has led to discrepancies between physicians and zoologists in defining what a cardiac outflow tract valve is. The aim here is to compare the gross anatomy of the outflow tract valvular system among several groups of vertebrates in order to understand the conceptual and nomenclature controversies in the field.This work was supported by Grant CGL2017-85090-P (Ministerio de Ciencia e Innovación, Madrid, Spain) and FEDER funds. M. T. Soto-Navarrete was recipient of fellowship PRE2018-083176 (Ministerio de Ciencia e Innovación, Madrid, Spain). M.A. López-Unzu was recipient of a Postdoctoral Fellowship from University of Málaga.S

Health-related quality of life among smoking relapsers

Background: Previous studies have shown that smoking is associated with health-related quality of life (HRQoL) impairment. In order to evaluate HRQoL in a sample of Spanish relapsers, a cross-sectional study was conducted. Method: The sample was made up of 775 smokers who had relapsed after a period of abstinence. HRQoL was evaluated using the Euro-Qol questionnaire (EQ-5D); through the descriptive profile, the EQ-5D index and the visual analogue scale (EQ-VAS). Results: Higher nicotine dependence was related to worse HRQL. According to the EQ-VAS, higher daily cigarette consumption and more years smoking were related to worse perceived health. In the EQ-5D those who had quit smoking in the previous year perceived worse health. Mobility and anxiety/ depression are the dimensions affected by smoking. Those who are more nicotine dependent (OR = 2.29) and have been smoking for longer (OR = 4.12) are more likely to have mobility problems; and those who are nicotine dependent (OR = 1.85) and relapsed more than a year ago (OR = 0.63), are more likely to experience anxiety/ depression. Conclusions: Nicotine dependence demonstrated a determining effect on HRQOL deterioration in smokers who have relapsed

Antifungal activity against Fusarium culmorum of stevioside, Silybum marianum seed extracts, and their conjugate complexes

Fusarium head blight (FHB) is a disease that poses a major challenge in cereal production that has important food and feed safety implications due to trichothecene contamination. In this study, the effect of stevioside—a glycoside found in the leaves of candyleaf (Stevia rebaudiana Bertoni)—was evaluated in vitro against Fusarium culmorum (W.G. Smith) Sacc., alone and in combination (in a 1:1 molar ratio) with polyphenols obtained from milk thistle seeds (Silybum marianum (L.) Gaertn). Different concentrations, ranging from 32 to 512 µg·mL−1, were assayed, finding EC50 and EC90 inhibitory concentrations of 156 and 221 µg·mL−1, respectively, for the treatment based only on stevioside, and EC50 and EC90 values of 123 and 160 µg·mL−1, respectively, for the treatment based on the stevioside–polyphenol conjugate complexes. Colony formation inhibition results were consistent, reaching full inhibition at 256 µg·mL−1. Given that synergistic behavior was observed for this latter formulation (SF = 1.43, according to Wadley’s method), it was further assessed for grain protection at storage, mostly directed against mycotoxin contamination caused by the aforementioned phytopathogen, confirming that it could inhibit fungal growth and avoid trichothecene contamination. Moreover, seed tests showed that the treatment did not affect the percentage of germination, and it resulted in a lower incidence of root rot caused by the pathogen in Kamut and winter wheat seedlings. Hence, the application of these stevioside–S. marianum seed extract conjugate complexes may be put forward as a promising and environmentally friendly treatment for the protection of cereal crops and stored grain against FHB