Embryological Evidence for the Formation of a Quadricuspid Aortic Valve in the Syrian Hamster
Borja Fern~adez, BSc* Ana C. DurS.n, PhD, t Gaetano Thiene, MD, t Manuel Cardo, BSc*
Josep M. Arqu6, MD,* and Valentfn Sans-Coma, PhD*
*Department of Animal Biology, University of Mdlaga, M6laga, Spain, t institute of Pathological Anatomy, University of Padua, Padua, Italy, and ~Regional Hospital "Carlos Haya," M61aga, Spain
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Congenital quadricuspid aortic valve is a rare anomaly, the morphogenesis of which remains unclear. In this study we report the case of a Syrian hamster embryo that presented an aortic valve with four valve cushions instead of three. The embryo, aged 12 days and 2 hours postcoitum, was at an early stage of valvulogenesis. It was examined using semithin sections of the heart. Two of the four valve cushions were located, one in the dorsal and the other in the left-ventral position, corresponding, respectively, to the dorsal and left valve cushions of a normal aortic valve. The remaining two cushions were situated in the right-ventral position. They were joined at the level of their basal portions and were less developed in size than the other cushions. This report seems to be the first embryological evidence for the formation of a quadricuspid aortic valve. The present findings strongly suggest that the anomalous valve originated from three mesenchymal ardagen and that the supernumerary valve cushion resulted from the division of the anlagen that normally gives rise to the right valve cushion.
Occurrence of a congenital quadricuspid aortic valve (QAV) is a very rare event (1-4), with an estimated frequency that ranges between 0.008% and 0.043 % (5-8). The' mechanism involved in the formation of QAVs is still unclear (9). The hy- potheses on this subject rely on the review of the gross anat- omy and pathologic findings in adults. To our knowledge, how- ever, no embryological evidence has currently been reported.
The Syrian hamster provides an animal model for inves- tigating the causes of spontaneous anomalies of the cardiac semilunar valves (10,11). In an ongoing study of the forma- tion of the aortic valve in this rodent species, an embryo was detected to have a QAV. We report this finding, believing that it may contribute to the understanding of the morphogenesis of this cardiac defect in humans.
Manuscript received April 4, 1994; accepted June 30, 1994. Supported by grants PB89-0577 and PB92-0413 from the DGICYT
(Ministerio de Educaei6n y Ciencia, Spain), the Dolores Platero Heart Fund- Comunidad de Propietarios "El Capistrano Villages and Acci6n Integrada Hispano-Itaiiana. Borja Fern~dez, Aria C. Dur'~, and Manuel Cardo are the recipients of fellowships FP92-33360070, EX92-25046339, and FP90- 34011113, respectively, from the Ministerio de Educaei6n y Cieneia, Spain.
Address for reprints: Borja Fem~indez, BSe, Department of Animal Bi- ology, Faculty of Science, University of M~llaga, E-29071, M~llaga, Spain. Tel: 34 5 2131853; Fax: 34 5 2132000
Materials and Methods The specimen that presented the QAV was detected among
a series of Syrian hamster embryos obtained from females belonging to a breeding colony in our laboratory. The ham- sters used in our research program are housed in polypropyl- ene cages in a room in which both the temperature and pho- toperiod are controlled. There is no known exposure of the animals to teratogenic agents. All hamsters are handled in compliance with the international policies of animal care and welfare. Pregnant females are killed by overdose with chlo- roform, and embryos are obtained by laparotomy and uterotomy. Thereafter, they are freed from fetal membranes, and their total length (TL) is measured. The age of the em- bryos is estimated by considering the coitus as day 0, hour 0.
The embryos were perfusion-fixed with 1% formaldehyde and 2 % glutaraldehyde in 0.05M sodium cacodylate buffer, with osmolarity adjusted to 330 milliosmol/liter. After sub- sequent immersion fixation in this fixative for 45 minutes, the embryos were washed with the buffer, postfixed in 1% os- mium tetroxide for 11/2 hours, and washed again. Removed hearts were dehydrated in acetone and embedded in araldite. Sagittal sections, serially cut at 1/zm with an ultramicrotome Reichert UMO-2, were stained with 1% toluidine blue in bo-
288 FERNANDEZ ET AL. Cardiovasc Pathol Vol. 3, No. 4 F O R M A T I O N O F Q U A D R I C U S P I D A O R T I C V A L V E O c t o b e r - D e c e m b e r 1 9 9 4 : 2 8 7 - 2 9 1
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Figure 1. Serial sections (A-D) cut in dorsoventral orientation of the left cardiac outflow tract of a Syrian hamster embryo (E-33.2), aged 12 days and 2 hours postcoitum (TL = 12 mm). The aortic valve had developed normally. Three valve cushions-dorsal (D), right (R), and left (L) -can be rec- ognized at the base of the aorta. (Toluidine blue; scale bar = 150 #m.)
Figure 2. Serial sections (A-H) cut in dorsoventral orientation of the left cardiac outflow tract of a Syrian hamster embryo (E-33.6), aged 12 days and 2 hours postcoitum (TL = 12 mm). The aortic valve shows four valve cushions: dorsal (D), right-1 (R1), right-2 (R2), and left (L). (Toluidine blue; scale bar = 150 #m.)
Cardiovasc Pathol Vol. 3, No. 4 FERNANDEZ ET AL. 289 October-December 1994:287-291 FORMATION OF QUADRICUSPID AORTIC VALVE
rax. Observations were made using a light microscope Nikon Microphot FXA.
Results The embryo with the QAV was aged 12 days and 2 hours
postcoitum (TL = 12 ram). For a clear presentation of this case, we will first describe the features of an aortic valve de- veloping normally, as observed in another embryo (TL = 12 ram) belonging to the same litter.
In the embryonic heart with a normal (tricuspid) aortic valve (Figs. 1A-1D) the septation of the conotruncus had al- ready taken place, and the tertiary foramen was still open (Fig. 1A). The dorsal (Figs. 1A and 1B), right, and left (Figs. 1C and 1D) aortic valve cushions (or cusps) could be clearly identified at the base of the aorta. They developed as three separate structures and were located in the dorsal, right- ventral, and left-ventral positions, respectively. Each cush- ion consisted of a mesenchymal core covered by endothelium and showed a mild degree of excavation.
In the embryonic heart with the QAV (Figs. 2A-2H) the septation of the conotruncus had concluded, and the tertiary foramen remained open. The aortic valve showed four valve cushions, each composed of mesenchymal tissue covered by endothelium. Two of the four cushions were of nearly equal size and showed a degree of excavation similar to that of the valve cushions in the normal embryonic heart. One was lo- cated in the dorsal (Figs. 2A-2C) and the other in the left- ventral (Figs. 2C-2H) position, thus corresponding, respec- tively, to the dorsal and left valve cushions of a normal aortic valve. The remaining two cushions were less developed in size and slightly excavated. They were situated in the right-ventral position and will be called right-1 and right-2 (Figs. 2D-2H): right-1 is adjacent to the dorsal cushion, and right-2 is adja- cent to the left cushion. At the level of their basal portions, the right-1 and right-2 cushions were joined to each other,
sharing a certain amount of mesenchymal tissue (Figs. 2G and 2H; 3A and 3B). Moreover, they remained slightly con- nected along their adjacent faces (Figs. 2H and 3B).
Finally, it should be noted that the embryo possessing a QAV showed a normal (tricuspid) pulmonary valve.
Discussion The normal development of the cardiac semilunar valves
does not substantially differ between humans (12,13) and non- human mammals (14,15). The primordia of the right and left leaflets of both the aortic and pulmonary valves form by the growth of the edges of the two mesenchymal swellings, the conotruncal ridges that fuse, dividing the lumen of the conotruncus into the aortic and pulmonary tracts. The primor- dia of the dorsal aortic valve leaflet and ventral pulmonary valve leaflet develop from the mesenchymal tissue of the dor- sal and ventral walls of the truncus, respectively. After the septation of the conotruncus, each developing cardiac semi- lunar valve shows three leaflet primordia or valve cushions. Each of them appears as an elongated swelling and is com- posed of a mesenchymal core covered by the endothelium. The process by which the aortic and pulmonary valve leaflets develop from the valve cushions to reach their definite semi- lunar shape is usually termed excavation. However, the mor- phogenetic mechanism by which the excavation takes place in mammals is still unclear (15).
The formation of QAVs remains a question. Abnormal mesenchymal proliferations in the common trunk have been suggested as producing such anomalous valves. The interpo- lation of an extra swelling or pad before the contruncal septa- tion would lead to the formation of the fourth leaflet (6,7,16). Anomalous septation of the conotruncus has also been ad- duced as a possible cause of the development of QAVs. Alter- ations in the formation of the aorticopulmonary septum might be responsible for the asymmetric arrangement of the cono-
Figure 3. (A and B) Enlarged portions of Figures 2G and 2H, respectively. Note that the right-1 (R1) and right-2 (R2) valve cushions are joined to each other at the level of their basal portions (A, B) and remain slightly connected along their adjacent faces (B). (Scale bar = 50/~m.)
290 FERN.~NDEZ ET AL. Cardiovasc Pathol Vol. 3, No, 4 FORMATION OF QUADRICUSPID AORTIC VALVE October-December 1994:287-291
truncal swellings, resulting in four aortic valve and two pul- monary valve cushions (6,7,16-19). On the other hand, it has been stated that the quadricuspid condition of the aortic valve might be caused by the anomalous excavation of one of the valve cushions. This abnormal hollowing-out process would divide one of the cushions into two (6). The supernumerary aortic valve leaflet might also result from the septation of a normal valve cushion because of an inflammatory epi- sode (20).
The present report seems to be the first embryological evi- dence for the formation of an aortic valve with four leaflets. The affected Syrian hamster embryo, aged 12 days and 2 hours postcoitum, was at a relatively early stage of valvulogenesis. Four aortic valve cushions (dorsal, left, right-l, and right-2) could be clearly recognized. Each cushion showed a mild de- gree of excavation. This strongly casts doubt on the hypothe- sis that the formation of the right-1 and right-2 cushions might result from the anomalous excavation of a single right cush- ion. It seems more likely that the aortic valve had formed as a quadricuspid structure from the onset. The possibility that a pathologic process is responsible for the development of the supernumerary cushion can also be ruled out, given that no signs of inflammatory reaction were observed.
In the affected embryo there was no evidence of anoma- lous septation of the conotruncus. Moreover, the specimen showed three normal pulmonary valve cushions and not a bi- cuspid pulmonary valve. Thus alterations in the formation of the aorticopulmonary septum can be excluded as a possible cause of the anomalous valve.
The present observations do not rule out the hypothesis that the quadricuspid condition of the valve may be attribut- able to the existence of four mesenchymal anlagen. The su- pernumerary cushion might have resulted from the interpo- lation of an extra swelling in the common trunk. In such a case, however, the extra swelling should have partially fused with the dextrodorsal conotruncal ridge. Indeed, the right-1 and right-2 valve cushions showed a common basal region and were joined along their adjacent faces.
Another possibility is that the present anomalous aortic valve had developed from three mesenchymal anlagen, not four. The dorsal and left valve cushions would have originated from the dorsal and left-ventral anlagen, respectively, whereas both the right-1 and right-2 cushions would have formed from the right-ventral artlagen. The features of the two right cushions tend to support this hypothesis. In addition to their connec- tion at the basal level, they were located where a single right valve cushion normally exists. Moreover, they were smaller in size than the dorsal and left valve cushions. These findings strongly suggest that the two right cushions had originated from the division of the aortic portion of the dextrodorsal mesenchymal swelling. This may be attributable either to the defective growth of the edge of the swelling, giving rise to a bilobed structure, or to the partition of the edge by invagi- nation of the endothelial layer. Whatever the mechanism of
division, it remains unclear whether the division takes place before, during, or just after the conotruncal septation. Fur- ther observation in embryos belonging to earlier developmen- tal stages is needed to clarify these questions.
We cannot assume that the causes of spontaneous anoma- lies in the Syrian hamster are identical to those in humans. Despite this caveat, our observations substantiate the exis- tence of a mechanism of formation of QAVs that has been over- looked until now. This mechanism does not involve anoma- lous mesenchymal proliferations in the common trunk, abnormalities in the contruncal septation, or defective exca- vation of a valve cushion. It relies on the early division of one of the three mesenchymal aortic valve primordia. In this context, it should be noted that de Vries (5) already mentioned the possibility that a quadricuspid cardiac semilunar valve may result from the division of one of the leaflets into two. How- ever, he gave no explanation of how this division occurs.
References 1. McRonald RE, Dean DC. Congenital quadricuspid aortic valve. Am
J Cardiol 1966;18:761-762.
2. Falcone WM, Roberts WC, Morrow AG, PerloffJK. Congenital aor- tic stenosis resulting from a unicommissural valve: clinical and ana- tomic features in twenty-one adult patients. Circulation 1971;44: 272-280.
3. Davia JE, Fenoglio JJ, DeCastro CM, McAllister HA, Cheiflin MD. Quadricuspid semilunar valves. Chest 1977;72:186-189.
4. James KB, Centorbi LK, Novoa R. Quadricuspid aortic valve: case report and review of literature. Texas Heart Inst J 1991;18:141-143.
5. de Vries WM. Ueber Abweichungen in der Zahl der Semilunarklap- pen. Beitr Pathol Anat 1918;64:39-54.
6. Simonds JP. Congenital malformations of the aortic and pulmonary valves. Am J Med Sci 1923;166:584-595.
7. Hurwitz LE, Roberts WC. ~Jadricuspid semilunar valve. Am J Cardioi 1973;31:623-626.
8. Feldman BJ, Khandheria BK, Warnes CA, Seward JB, Taylor CL, Ta- jik AJ. Incidence, description and functional assessment of isolated quad- ricuspid aortic valves. Am J Cardiol 1990;65:937-938.
9. Brouwer MHJ, de Graaf JJ, Ebels T. Congenital quadricuspid aortic valve. Int J Cardiol 1993;38:196-198.
10. Sans-Coma V, Cardo M, Thiene G, Fernftmiez B, Arqu6 JM, Dunin AC. Bicuspid aortic and pulmonary valves in the Syrian hamster. Int J Cardiol 1992;34:249-254.
11. Sans-Coma V, Cardo M, Dur,~n AC, Franco D, Fermindez B, Arqu6 JM. Evidence for a quantitative genetic influence on the formation of aortic valves with two leaflets in the Syrian hamster. Cardiol Young 1993;3:132-140.
12. Kramer TC. The partitioning of the truncus and conus and the forma- tion of the membranous portion of the interventricular septum in the human heart. Am J Cardiol 1963;71:343-370.
13. Maron BJ, Hutchins GV. The development of the semilunar valves in the htmmn heart. Am J Pathol 1974;74:331-344.
14. Shaner RF. Abnormal pulmonary and aortic semilunar valves in em- bryos. Anat Rec 1963;143:5-13.
15. Hurle JM, Colv~e E, Blanco AM. Development of mouse semilunar valves. Anat Embryol 1980;160:83-91.
16. Coeurderoy A, Biron Y, Lanrent M, Almagne C. Quadrieuspidie aor- tique cong6nitale. Apropos dun cas. Revue de la litt~rature. Arch Mal Coeur 1986;5:745-748.
Cardiovasc Pathol Vol. 3, No. 4 FERNANDEZ ET AL. 291 October-December 1994:287-291 FORMATION OF QUADRICUSPID AORTIC VALVE
17. Peretz DI, Changfoot GH, Gourlay RH. Four-cusped aortic valve with significant hemodynamic abnormality. Am J Cardiol 1969;23:291-293.
19. Wyatt JP, Goldenberg H. Supernumerary aortic cusps with multiple
fenestrations and with displacement of the left coronary orifice. Arch Pathol 1948;45:784-786.
20. Salvatore I, Mincione G, Baldelli P. Valvola aortica quadricuspide con insufficienza aortica. Un caso diagnosticato angiograficamente ed oper- ato. G Ital Cardiol 1976;6:323-326.
