Pulmonary ductal coarctation and left pulmonary artery interruption; pathology and role of neural crest and second heart field during development


Gittenberger-de Groot AC, Peterson JC, Wisse LJ, Roest AAW, Poelmann RE, Bökenkamp R, Elzenga NJ, Hazekamp M, Bartelings MM, Jongbloed MRM, DeRuiter MC.PLoS One. 2020 May 15;15(5):e0228478. doi: 10.1371/journal.pone.0228478. eCollection 2020.PMID: 32413023 Free PMC article



Objectives: In congenital heart malformations with pulmonary stenosis to atresia an abnormal lateral ductus arteriosus to left pulmonary artery connection can lead to a localised narrowing (pulmonary ductal coarctation) or even interruption We investigated embryonic remodelling and pathogenesis of this area.

Material and methods: Normal development was studied in WntCre reporter mice (E10.0-12.5) for neural crest cells and Nkx2.5 immunostaining for second heart field cells. Data were compared to stage matched human embryos and a VEGF120/120 mutant mouse strain developing pulmonary atresia.

Results: Normal mouse and human embryos showed that the mid-pharyngeal endothelial plexus, connected side-ways to the 6th pharyngeal arch artery. The ventral segment formed the proximal pulmonary artery. The dorsal segment (future DA) was solely surrounded by neural crest cells. The ventral segment had a dual outer lining with neural crest and second heart field cells, while the distal pulmonary artery was covered by none of these cells. The asymmetric contribution of second heart field to the future pulmonary trunk on the left side of the aortic sac (so-called pulmonary push) was evident. The ventral segment became incorporated into the pulmonary trunk leading to a separate connection of the left and right pulmonary arteries. The VEGF120/120 embryos showed a stunted pulmonary push and a variety of vascular anomalies.

Summary: Side-way connection of the DA to the left pulmonary artery is a congenital anomaly. The primary problem is a stunted development of the pulmonary push leading to pulmonary stenosis/atresia and a subsequent lack of proper incorporation of the ventral segment into the aortic sac. Clinically, the aberrant smooth muscle tissue of the ductus arteriosus should be addressed to prohibit development of severe pulmonary ductal coarctation or even interruption of the left pulmonary artery.




Fig 1 a. Schematic view of the normal perinatal aortic arch showing the ascending aorta (aAo) and the pulmonary trunk (PT). De ductus arteriosus (DA) connects the PT with the descending aorta (dAo). The left (lpa) and right (rpa) pulmonary arteries are dorsally hooked up to the PT. Fig 1b. Schematic representation of a case with an abnormal lateral DA to lpa connection creating a proximal (plpa) and distal (dlpa) part. Aberrant DA tissue (black indentations), extending into the plpa, create the narrowing hallmark of the pulmonary ductal coarctation.


Fig 2 a. Schematic representation of a dorsal view of the connection of the vasculature of the mid-pharyngeal endothelial strand (mpes) and connections to the combined ventral sprouts (v4/6) of the right and left 4th and 6th PAA continuous with the aortic sac (AoS) in an E10.0 WntCre mouse embryo. The dorsal sprouts of 4th and 6th PAA (d4 and d6) are connected to the dorsal aorta (indicated is the left one, ldAo). Fig 2b, 2c and 2d: Reconstructions from various angles of relevant structures showing the WntCre positive neural crest cells (olive green) surrounding the continuous PAA arteries (1,2,3) as well as the AoS (out of view in b.) The dorsal aortae are on their lateral and dorsal aspect surrounded by NCC. The myocardium is depicted in shades of grey. The mid-line mesenchymal second heart field mass (SHF: yellow) is positioned between the confluence of the 3rd (running anteriorly over the AoS; blue arrow in c) and the developing 4th and 6th PAAs (in b, c). Fig 2d. In this reconstruction the mid-line SHF mesenchymal mass has been removed showing the extent of coverage of the cranial part of the mpes (orange) while more caudally the mpes is not surrounded by SHF (pink). Fig 2e. transverse section at the level (see black line in d) of the connection (white arrow) of the cranial part of the mpes (white lines) and endothelial cells (stained red for CD31) to the left v4/6 (NCC: green). The NKx2.5 positive SHF cells (yellow) border this connection and surround the endothelial cells here in the midline. Magnification: bar 100 μm.


Fig 3 a. Schematic representation of a dorsal view of vascular connections in an E11.5 WntCre mouse embryo. In this stage PAA 3, 4 and 6 are complete. The distal left (dlpa) and right (drpa) pulmonary arteries merge sideways with PAA 6 creating a ventral (v6) and a dorsal (d6) segment of the latter. The d6 is completely encircled by neural crest cells (green) while the v6 has a lateral layer of neural crest and a medial layer of second heart field (SHF: yellow). The v6 forms a short proximal pulmonary artery. Fig 3b. dorsal view of a reconstruction at this stage with now complete 3rd,4th and 6th arches that are lined by neural crest (olive green). The SHF (yellow) forms the mid-line mesenchyme. The dlpa and drpa (pink) are not covered by either NCC or SHF. Fig 3c. left lateral view showing the connection of the outflow tract (OFT) myocardium (grey) with the short aortic sac (AoS). The SHF mass has a short extension anteriorly towards the future aortic orifice (blue arrow). More prominent at this stage is the left sided SHF extension that runs underneath the v6 and along the future pulmonary wall of the AoS. This is the so-called pulmonary push (PP). Fig 3d. dorsal view after removal of the SHF, the double lining of the relatively short v6 (identical to a proximal part of a pulmonary artery (see also a.) is depicted with a NCC (olive green) and a SHF reflected coverage (orange) Fig 3e. section of the embryo at the level indicated (black line) in d. The right and left v6 (endothelial cells red) are in part lined by NCCs (green) and Nkx2.5 positive SHF (yellow). In the SHF midline mass (yellow) the endothelial plexus (red) of the mid-pharyngeal endothelial strand (mpes) is visible. Magnification: bar 100 μm.



Fig 4 a and f schematic representations of two E 12.5 WntCre embryos. Embryo a. is slightly less far developed as compared to f. In both embryos the ascending aorta (aAo) and the pulmonary trunk (PT) have now been separated. Fig 4b-4d shows reconstructions of the younger embryo with a complete 4th and 6th PAA. Fig 4c shows the dorsal midline SHF mass (yellow) partly covering the neural crest (green) lined PAA’s. Fig 4c is a left-sided view showing the position of the SHF mass (yellow) which has an extension (pulmonary push population: PP) covering the major part of the lumen of the PT towards the myocardial outflow tract (grey), running underneath the 6th PAA to pulmonary artery connection. Fig 4d. dorsal view after removal of the SHF mass (lumen coverage area: orange). On the left side the dorsal 6th PAA, now referred to as ductus arteriosus (DA) is completely surrounded by neural crest cells, There is no indication of a ventral 6th PAA. The left pulmonary artery (lpa: red) abuts independently on the PT On the right side the situation is less well developed. The dorsal segment of the right 6th PAA (rd6) is regressing and completely surrounded by NCC. The ventral segment of the 6th PAA (rv6) is still present with a lateral wall of NCCs and a medial wall of SHF. The distal part of the right pulmonary artery (drpa) enters side-ways into this right-sided 6th PAA. Thus the proximal rpa is at this stage formed by the rv6. Fig 4e. section at the level indicated in d (black line) in which it can be seen that the right v6 (rv6: also proximal part of the rpa) has both a lining of SHF (yellow) and NCCs (green). Fig 4f in the more developed embryo the originally distal parts of the lpa and rpa, embedded in Mef2c positive mesoderm (Fig 4k) are not covered by NCC and SHF. They enter the PT independent of the DA. Fig 4g-4i are reconstructions showing similar dorsal views as b-d. Level of section j. is indicated by a black line in i. The SHF derived flow divider is still seen between the lpa and rpa (blue arrows in g and i and white arrow in j). The right d6 has regressed completely. There is on both sides no indication anymore of the v6 segments. k section of a E12.5 Mef2cCre embryo showing that the distal parts of the lpa and rpa (white dotted vessels) are situated within Mef2c positive splanchnic mesoderm that is not stained by Nkx2.5 (green). T: trachea Magnification sections e,j,k bars100 μm.

Fig 5 Human embryos in an upclimbing age series, Reconstructions of embryos depicted in a,b,c have been stained with hematoxylin eosin. a. Reconstruction of a very early human embryo (4 mm, CS 12) with a complete 3rd PAA. The combined ventral sprouts of the 4th and 6th PAA are in contact with the mid- pharyngeal endothelial plexus (mpes) all depicted in grey and surrounding the developing gut (light green, G). Fig 5b, 5c left lateral and dorsal view of a lumen reconstruction of a human embryo (6.5 mm, CS14) with complete 3rd and 4th arches (3,4). The dorsal 6th artery (d6) is indicated in green, the myocardium of the outflow tract in grey and the distal pulmonary arteries in red. The dorsal view the short ventral segments of the 6th PAAs (v6) form a short proximal pulmonary artery. The distal part of the pulmonary arteries (dlpa,drpa:red) are thus in contact with the AoS. Fig 5d, 5e. sections of a human embryo (CS16) which is stained for neural crest for TFAP2α (green) which clearly surrounds the ld6/DA (d). Fig e. There is a marked extension of Nkx2.5 positive SHF (yellow) along the outer side of the PT (pulmonary push population: PP). It cannot be discerned whether the lpa and rpa (white dotted rings) are of proximal or distal order. Fig 5f-5g reconstruction of an older human embryo (17mm, CS19) stained for HNK1. The ascending aorta (aAo) and the PT have been separated. The right 6th PAA has disappeared. The lpa and rpa (pink) are connected separately to the dorsal side of the PT, sharing the HNK1 antibody staining (purple) that allows for distinguishing boundaries between the left sided dorsal 6th PAA (ductus arteriosus: DA, green) and the PT (light blue). The DA continues smoothly into the PT with a more anterior position as compared to the lpa and rpa. Fig 5h, 5i. transverse sections (see section levels as black lines in f and g) of the HNK1 stained pulmonary arteries (lpa and rpa) and the connection of the rpa to the PT, while the main part of the PT and the DA are negative for this staining. Abbreviations: n.X vagal nerve, T trachea. Magnification: bars 100 μm.


Fig 6 a. Angio of a patient with pulmonary atresia without VSD in which the leftsided DA (asterisk) is positioned above and anterior of the left pulmonary artery. The left (lpa) and right (rpa) pulmonary arteries (venous line: blue arrow; arterial line yellow arrow) are not compromised at their origin. Fig 6b. Morphology of the arterial pole of a heart specimen in which the ductus arteriosus (DA) connects anteriorly to the pulmonary trunk (PT), while the rpa and lpa are more dorsally connected. Fig 6c. Angio of a patient with tetralogy of Fallot with a marked narrowing (blue arrow) of the proximal (origin) left pulmonary artery (DA dotted area). Fig 6d Postmortem specimen with atresia of the PT and a confluence of the rpa and lpa, with a smaller diameter of the latter. The DA enters sideways into the lpa which shows a PDC at the site of connection (blue arrow). Fig 6e. sagittal section of a human fetal (resorcine fuchsine stained for elastin). The elastin poor muscular DA only lined on the lumen side by an internal elastic lamina, connects with a fishtail like construction (arrows) to the elastin rich rpa and lpa. No DA tissue is encountered in the wall of the lpa and rpa. Fig 6f. Sagittal section (resorcine fuchsin stained for elastin) of a specimen with severe pulmonary ductal coarctation. The tissue of the elastin poor DA, presenting with intimal cushions (arrowheads), is inserted sideways in the wall of the plpa (asterisk), while the dlpa is still elastic in nature. Fig 6g. detail of f in a subsequent section stained for Azan in which it can be seen that the elastic lamellar structure (yellowish) of the dlpa and plpa is interrupted by the adventitia (dotted area) of the DA. Fig 6h. 2D echocardiographic image of the patient with DORV on prostaglandin. In this high parasternal short axis view the rpa, lpa and DA (dotted area) are indicated. The arrow points to the lpa origin. Fig 6i. Same image with Doppler color showing the flow to the rpa and lpa in blue and the DA flow in orange and green. Fig 6j. 2D echo of the same patient 10 days later after placement of the right mBT shunt and discontinuation of prostaglandin. Same view as Fig 6h. The DA has closed. The origin of the lpa is severely stenosed marked by the arrow. Fig 6k. same image as i with showing the flow to the rpa. Because of the mBTS the rpa the flow is turbulent coded in green. The lpa does not receive flow because of the severe stenosis at its origin. The DA has closed. Magnification: e-g bars: 100 μm.


Fig 7 Reconstruction (left lateral views) of a E10.5 VEGF WT (a) and a VEGF 120/120 (b) mouse embryo in which the SHF has been stained for Nkx2.5 (yellow). There is a marked difference in the pulmonary push (PP) which extends along the future pulmonary side of the aortic sac (AoS) for quite some distance in the WT embryo (a), while in the mutant embryo (b) the PP is stunted. In both embryos the lpa and rpa connect to the AoS by way of ventral sprout of the 6th PAA (v6) and are embedded in the SHF tissue. Fig 7c, 7d reconstructions of an E14.5 mutant embryo in which the hypoplastic DA (green), arising vertically from the aortic arch, is connected side-ways to the lpa (red). There is atresia of the pulmonary orifice and the pulmonary trunk (PT) is hypoplastic. Fig 7e-7h Sections stained for ɑ smooth muscle actin (1A4) of the site of connection of the lpa and rpa into the PT. e and f. are E15.5 embryos in which (e) the WT shows equal sizes of the lpa and rpa with a small posterior separating ridge (open arrowhead), while in the mutant embryo (f) the origin of the lpa, which is relative smaller in diameter as compared to the rpa, is flanked by thickened DA tissue (arrows). In a WT (g) and mutant embryo (h) of E 17.5 we observe a similar phenomenon although in the mutant embryo (h) the small lpa, encircled by thick ductal tissue (arrows) is connected to a right-sided DA (rDA) continuing into a right aortic arch (rAoA). Magnification bars in e-h 100 μm.