5.1 INTRODUCTION
The results from the previous chapters have provided evidence for the presence of both cell-surface metalloendopeptidases during a period in the development of the rat embryo when a considerable degree of morphogenesis is taking place in the craniofacial region. However, the presence of these enzymes does not provide any information about the roles that they play during embryogenesis. One way of investigating the possible roles played by these enzymes during post-implantation development in the rat, is to culture whole embryos in the presence of selective inhibitors. Over the past decade a range of selective inhibitors of NEP have been developed (reviewed by Wilkins et al., 1993).
The therapeutic consequences of inhibition of NEP have focused on the potentiation of physiological peptide substrates in an attempt to treat serious diseases including hypertension and congestive heart disease. The peptide substrates involved in these conditions include ANP, bradykinin and endothelin-1. It has also been proposed that the potentiation of the enkephalins and endorphins following inhibition of endogenous NEP could form part of an effective analgesic therapy (Wilkins et al., 1993). The substrates found in the respiratory system such as substance P, have been demonstrated
to play a significant role in airway inflammation and asthma, and one potential therapy currently being developed involves local application of recombinant NEP (personal communication, Dr D. Brennen, Khepri Pharmaceutical, Inc., San Francisco, USA).
The finding that NEP is identical to CALLA (Shipp et al., 1989) which has been found in solid tumours (such as small cell carcinomas of the lung, Shipp et ah, 1991, and, hepatocarcinoma, Dragovié et al., 1994) as well as on the leukaemic blast cells, suggests an avenue for cancer therapy, possibly by providing a target for tumouricidal drugs.
To date no specific inhibitors directed against Endo-2 have been described. The most selective is actinonin, but as this inhibits other metallo-peptidases at low concentrations it was not used in this study.
However, advantage has been taken of the availability of two highly selective inhibitors of NEP, phosphoramidon, a natural microbial product, and the other, thiorphan a synthetic drug, to establish whether or not rat embryos cultured in the presence of these inhibitors would undergo normal development. If, as has been postulated, NEP plays an important role during normal craniofacial development, then inhibition of NEP should result in abnormal development.
5.2 MATERIALS AND METHODS
Rat embryos at E9.5 (head-fold stage) and E10.5 were dissected from pregnant Wistar rats under a still-air hood as quickly as possible, and cultured with their yolk sac and amnion intact for 45-48 hr according to protocols described by Cockcroft, 1990. The dissection and culture procedures, including the gassing regime, are detailed above (2.1.2 and 2.18). The culture medium used was immediately-centrifuged rat serum (Cockcroft,
1990) diluted in sterile Hank’s buffered saline. Diluted rat serum was equilibrated with the first gas mixture prior to the start of the culture period, and warmed to the culture temperature (37°C) in the incubator. The two highly selective NEP inhibitors used in this study, phosphoramidon and thiorphan have been described previously (1.6.1 and 2.18.1).
Comparatively fewer experiments were carried out using thiorphan due to the possibility that the introduction of ethanol into the experiment might produce a vehicle effect, and therefore make the interpretation of the data more difficult. However, it was felt that some experiments with thiorphan had to be done in order to complement the phosphoramidon data, especially as it has been reported that endothelin-converting enzyme is also inhibited by high concentrations (> 5 0 ^M) of phosphoramidon. Endothelin-converting enzyme is not inhibited at all by thiorphan.
After culture, embryos were examined under the dissection microscope and scored using eight criteria of normality. After scoring, embryos were fixed and processed for wax histology and scanning electron microscopy as described above (1.3.2-3 and 2.4.2- 3).
5.3 RESULTS
The data presented below are the combined results from several culture experiments. The phosphoramidon cultures at both embryonic stages were each carried out on five separate occasions, and the thiorphan cultures on two separate occasions. The number of embryos cultured in each experiment was entirely dependent on the number of rats which were pregnant, the size of their litters, and the number of embryos at the correct stage at the onset of the culture period. The total number of embryos scored was 223, this number does not include any embryos that failed to develop.
F ig. 5A . S EM photograph showing a rat em bryo cultured for 48 hr, from B9.5, in im m ediately-centrifuged rat serum diluted in sterile H ank’s buffered saline only. This control em bryos does not exhibit any visible m orphological abnorm alities and was deem ed norm al according to the eight criteria o f norm ality used to score em bryos post-culture.
Bar = 200 m icrons.
F ig. 5B . SEM photograph showing a rat em bryo cultured for 48 hr, from E 9.5, in serum containing 10 nM phosphoram idon; note the failure o f crcuiial neural fold fusion (arrow ) over the developing forebrain region.
Bar = 1 0 0 m icrons.
F ig. 5 C . SEM photograph showing a rat em bryo cultured for 48 hr, from E 9.5, in serum containing 100 nM phosphoram idon; note the swelling (asterisk) on the left side o f the prosencephalon.
Fig. 5D. SEM photograph showing a rat embryo cultured for 48 hr, from E9.5, in serum containing 100 juM phosphoramidon; note the open cranial neural folds (N) and the swelling (arrowheads) of the prosencephalic neuroepithelium into the lumen, caused by a subadjacent haematoma.
Bar = 100 microns.
Fig. 5E. Lower magnification view of Fig. 5D. showing the disturbance to the normal looping morphogenesis of the heart (asterisk). Bar = 100 microns.
Fig. 5F. SEM photograph showing a rat embryo cultured for 45 hr, from E10.5, in immediately-centrifuged rat serum diluted in sterile Hank’s buffered saline only. This control embryos does not exhibit any visible morphological abnormalities and was deemed normal according to the eight criteria of normality used to score embryos post-culture.
Bar = 200 microns.
Fig. 5G. SEM photograph showing a rat embryo cultured for 45 hr, from E10.5, in serum containing 10 fiM phosphoramidon; note the relatively distended left side of the prosencephalon (asterisk) and the asymmetry of the first branchial arch.
Bar = 100 microns.
Fig. 5H. SEM photograph showing a rat embryo cultured for 45 hr, from E10.5, in serum containing 1 /xM phosphoramidon; note the grossly abnormal left side of the head with a swollen dysmorphic maxillary prominence (asterisk), and abnormal first and second branchial arch. Bar = 200 microns.
Fig. 51. SEM photograph showing a rat embryo cultured for 45 hr, from E10.5, in serum containing 100 fxM phosphoramidon; note the large spherical bulge (asterisk) on the maxillary prominence, just caudal to the eye primordium.
Figs. 5J-M. Haematoxylin and eosin stained coronal sections through a wax- embedded E9.5 rat embryo cultured for 48 hr in the presence of 1 /xM phosphoramidon. Bar = 20 microns.
Fig. 5J. Section through the forebrain. Note the greatly overgrown neuroectoderm (asterisk) on the left side of the prosencephalon.
Fig. 5K. More caudal section. Note the appearance of the left internal carotid artery (large arrow) compared to its contralateral partner (small arrow).
Fig. 5L. More caudal section. Note the now grossly dilated left internal carotid (large arrow) compared with the right (small arrow). The optic vesicle is also indicated (hollow arrow). The cranial neural folds have also failed to close.
Fig. 5M. Section at the level of the first branchial arch. Note the abnormally displaced left arch (arrow), the relatively distended first branchial arch artery (asterisk) and the open neural folds.
Immediately following the culture period the embryos were scored, and this data is presented below in tables 5.1-5.3. The control embryos from all of the culture experiments all appeared to have developed normally, an assessment based upon their gross morphological appearance both immediately post-culture and under the SEM, as well as their criteria of normality scores. A representative control embryo cultured for 48 hr from E9.5 is shown in Fig. 5A, whilst a control embryo cultured for 45 hr from E10.5 is shown in Fig. F. Of the thiorphan controls, the embryos cultured in diluted serum plus ethanol were grossly normal, although their crown-rump lengths, somite numbers and turning scores suggests that these embryos were slightly growth retarded when compared to the serum-only controls (Table 5.3).
Following culture in the presence of both phosphoramidon and thiorphan, SEM revealed that a considerable percentage of the inhibitor-treated embryos, cultured from E9.5 displayed an asymmetric, predominantly left-sided, facial deformity. This deformity appeared to have been caused by the presence of a haematoma-like swelling adjacent to the prosencephalon. Although subsequent histological examination revealed that the swelling was not due to a haematoma. This often appeared to have the effect of distending the left side of the head which in turn disrupted the normal positioning of the first and second branchial arches. Representative embryos are shown in Figs. B and C. In the most severe cases (shown in Figs. 5D and E), observed at the highest concentration of inhibitor (1(X) ^M), the swelling was so great that the cranial neural folds were appeared not to have been able to close.
Table 5.1. Results of phosphoramidon exposure on £9.5 rat embryos in vitro Inhibitor conc. No. of embryos Yolk sac diameter mm ± s.d. Crown- rump length mm ± s.d.
% Turned Somite No. ± s.d. % Heart beating % Yolk sac circulation % Cranio facial defect ^Severity of defect 0 (Control) 25 3.19 ± 0.24 3.06 ± 0.52 84 24.80 ± 1.79 100 100 0 - 10 nM 16 2.63 ± 0.25 2.35 ± 0.18 37.5 21.75 ± 0.5 100 100 25 + 100 nM 19 3.25 ± 0.20 2.88 ± 0.63 100 24.50 ± 0.58 100 100 26.3 + + 1 f i M 22 2.88 ± 0.14 3.06 ± 0.31 72.7 22.25 ± 2.22 72.7 72.7 45.4 + + 10 16 3.13 ± 0.25 2.94 ± 0.43 75 22.75 ± 0.96 100 100 25 + + + 100 f i M 12 3.17 ± 0.29 3.08 ± 0.14 100 23.67 ± 1.15 100 100 33 + + +
folds, clearly discernible haematoma and dysmorphic branchial arches).
Table 5.2. Results of phosphoramidon exposure on E10.5 rat embryos in vitro Inhibitor conc. No. of embryos Yolk sac diameter mm ± s.d. Crown-rump length mm ± s.d.
% Turned Somite No. ± s.d. % Heart beating % Yolk sac circulation % Cranio facial defect ♦Severity of defect 0 (Control) 11 4.43 ± 2.51 4.30 ± 2.00 100 31.16 ± 1.52 100 100 0 - 10 nM 27 4.10 ± 3.60 4.03 ± 3.78 100 27.33 ± 3.05 100 100 48.1 + 100 nM 23 4.20 ± 3.60 4.05 ± 3.01 100 28.33 ± 3.21 100 100 26 + 1 |iM 9 3.78 ± 4.91 3.36 ± 7.95 100 28.40 ± 4.39 77.7 55.5 77.7 + + 10 /*M 9 4.26 ± 0.57 4.30 ± 1.00 100 29.33 ± 1.15 100 100 33 + + 100 9 3.56 ± 8.14 3.4 ± 3.6 100 28.67 ± 1 .1 5 100 66.6 33 + +
folds, clearly discernible haematoma and dysmorphic branchial arches).
Table 5.3. Results of thiorphan exposure on E9.5 rat embryos in vitro Inhibitor conc. No. of embryos Yolk sac diameter mm ± s.d. Crown-rump length mm ± s.d.
% Turned Somite No. ± s.d. % Heart beating % Yolk sac circulation % Cranio facial defect ♦Severity of defect Serum only control 4 3.75 ± 0.44 3.30 ± 0.54 100 29.00 ± 1 .1 5 100 100 0 - Serum + ethanol control 3 3.80 ± 0.87 2.90 ± 0.36 33 27.00 ± 1.41 100 100 0 - 100 nM 6 1.85 ± 0.33 1.53 ± 0.38 33 17.83 ± 1.33 100 100 0 - 1 /xM 6 3.20 ± 0.62 3.06 ± 0.64 33 24.00 ± 1.73 50 50 33 + + 10 /xM 6 1.8 ± 0.44 1.63 ± 0.20 0 17.33 ± 1.15 50 50 33 + + +
folds, clearly discernible haematoma and dysmorphic branchial arches).
The severity of the defect, but not the incidence, generally increased as the concentration of the inhibitor was increased. In addition to these craniofacial effects, the heart appeared disproportionately swollen, in a midline position and seemed not to have undergone normal looping morphogenesis (Fig. 5E). Of the other parameters measured, there was only an extremely small decrease in crown-rump length, somite number, heart beat and yolk sac diameter, which suggests that the abnormalities observed in the inhibited embryos are specific effects of the inhibitors and not simply the result of poor culture technique.
Often inhibitor-treated embryos at the end of the culture period appeared almost normal when compared to the controls, but some of the more subtle manifestations of the characteristic defects could only be appreciated when viewed on the SEM. When E9.5 and E10.5 embryos were cultured in the presence of high concentrations ( > 1 fiM) of either of the inhibitors, a few embryos completely failed to develop.
The embryos which were at E10.5 at the start of the culture period also displayed an abnormal left-sided prosencephalic swelling, the extent of the abnormality appeared to increase with an increased inhibitor concentration (Figs. 5G-I). None of the E10.5 embryos had open cranial neural folds as they had closed prior to the onset of culture, but the branchial arches were often displaced asymmetrically and of an abnormal spatulate appearance. In contrast with the embryos cultured from E9.5, the older embryos exhibited grossly normal heart morphogenesis post-culture, based solely upon their appearance under SEM and the dissection microscope, although these embryos were not examined histologically.
Subsequent histological analysis of inhibitor-treated embryos cultured from E9.5, revealed that the prosencephalic swelling was the result of both a localised overgrowth
of, what appeared to be, the prosencephalic neuroectoderm on the affected side (Fig. 5J) and a gross distension of the internal carotid artery on the same side (Figs. 5K and L). However the endothelium of the vessel appeared to be intact. More caudally, this vascular disturbance extended to include the first branchial arch artery, which like the internal carotid, arises from the dorsal aorta, and displayed a considerable degree of distension. A presumed secondary effect of this, was the lateralward displacement of the first branchial arch (Fig. 5M).
A small number of embryos cultured in the presence of the highest concentrations of phosphoramidon and thiorphan completely failed to develop and died during the culture period. The possibility exists that the effects on these embryos were a direct consequence of the presence of the inhibitors, but as these conceptuses were extremely difficult to score, it was decided that, whilst this is an important finding which had to be reported, they would have to be excluded from the final analyses. This however, resulted in fewer inhibited embryos being available for post-culture scoring compared with controls.
5.4 DISCUSSION
The aim of the experiments described in this chapter was to determine if inhibition of NEP had any effect on the development of the E9.5 and E10.5 rat embryo in vitro.
Results from previous chapters have established the spatio-temporal distribution of NEP at both the protein and mRNA level in the post-implantation rat embryo across the gestational stages (i.e. from E9.5 to E12.5) used in the whole embryo culture experiments described in this chapter. Assessment of relative normality was based upon the following criteria: yolk sac diameter and circulation, crown-rump length, turning and the presence of a beating heart compared against control. These criteria were obtained
from similar published studies in which whole embryo culture was used to assess the effects of cysteine proteinase inhibitors on rat embryos (Ambroso and Harris, 1994).
The specific, apparently regionalised effects observed when embryos at both stages were cultured in the presence of selective inhibitors of NEP were not anticipated. The craniofacial vasculature, whilst immunohistochemically positive for NEP, was certainly not one of the strongest sites of NEP protein or mRNA distribution. Neither was the neuroectoderm in the region of the developing forebrain an especially rich source of the enzyme. Although the abnormalities in the craniofacial region were the most obvious when the cultured embryos were observed under the SEM, an adverse effect upon other developing organ systems cannot be ignored. The effects on the heart, for instance, merit further investigation in the future.
Under SEM, the open cranial neural folds, branchial arch deformities and the haematoma-like swelling were the most obvious manifestations of the consequences of inhibition. It was almost impossible to establish the physiological changes resulting in the swelling without sectioning these embryos. When the embryos were examined under the dissection microscope at the end of the culture period, many of the inhibited embryos at both stages had a clearly discernible red spherical swelling in the forebrain region which was always unilateral and almost always on the left-hand side. The appearance of this swelling indicated that it was most likely to be a localised haemorrhage (that is, a haematoma), or, a localised mass of blood vessels (a haemangioma) which had formed either de novo (vasculogenesis) or had sprouted from the established vasculature (angiogenesis).
In all of the whole embryo culture experiments the inhibitor was added at the start of the culture period. No attempts were made to assess the activity of the inhibitors used in this study at particular time points during the culture period. There are several ways in which this important question may be addressed in future experiments. One way would be to take an aliquot of the culture media at regular time-points and incubate it with purified NEP and a labelled NEP substrate. Any decrease in the rate that the substrate was broken down would indicate inhibitor activity. This approach could also be applied to samples of the contents of the yolk sac and amnion to confirm that the inhibitor had penetrated these extraembryonic membranes. Labelling the inhibitor with either a radionucleotide or fluorescent tag are further means by which the uptake of the inhibitor by the embryo may be followed.
Earlier studies in which the physiological roles played by NEP in the adult cardiovascular system have been investigated have identified peptide substrates of NEP which are implicitly involved in vasodilation and vasoconstriction. These include endothelin-1, bradykinin, angiotensin 1 and ANP. Indeed, much of the interest shown by the pharmaceutical industry in developing NEP inhibitors has centred on the control of hypertension. However there have been no reports of NEP being involved in any way with the formation of new blood vessels. An examination of the immunological and cancer-based studies failed to uncover a role for NEP in angiogenesis or vasculogenesis.
Therefore, it is possible that the defects observed under the SEM, reflected a secondary consequence of an abnormal rise in blood pressure, and not a dysmorphogenesis. Examination of the histological sections of inhibitor-treated embryos, however, indicated the contrary. As clearly shown in Fig. 5J, the neuroectoderm overlying the internal carotid artery appears to have grown abnormally dense around the
vascular lesion, significantly increasing the size of the swelling, to the extent that the cranial neural folds were unable to fully close. The nature of the lesion itself also became apparent. It was clear that the endothelial lining of the internal carotid had remained intact, and it appears that it had become considerably stretched. The artery itself though, was greatly dilated on the affected side and was packed full of blood cells. This would appear to rule out the idea that the swelling is a localized formation of new blood vessels.
If the vessel had simply ruptured, especially if it occurred early on in the culture period, the haemorrhaging would have been more widespread, and not confined within a spherical shell of endothelium. The first branchial arch artery on the same side as the prosencephalic lesion also appeared to be disproportionately dilated compared to its contralateral partner, and was also packed full of nucleated red blood cells. It is likely that this subsequently caused the abnormal distension of the first branchial arch.
The possible effects of inhibition on the other developing organ systems in the rat embryo shown in this study to be rich in NEP at both the mRNA and protein levels, were not examined histologically in order to remain focused on the development of the craniofacial region.
The results from these experiments suggest that NEP is essential for the