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Circulation Research. 1997;80:444-451

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(Circulation Research. 1997;80:444-451.)
© 1997 American Heart Association, Inc.

Articles

Embryonic Endothelial Cells Transdifferentiate Into Mesenchymal Cells Expressing Smooth Muscle Actins In Vivo and In Vitro

M.C. DeRuiter, R.E. Poelmann, J.C. VanMunsteren, V. Mironov, R.R. Markwald, , A.C. Gittenberger-de Groot

From the Department of Anatomy and Embryology (M.C.D., R.E.P., J.C.V., A.C.G.-de G.), Leiden University, Leiden, the Netherlands, and the Department of Cell Biology (V.M., R.R.M.), Medical University of South Carolina, Charleston.

Correspondence to M.C. DeRuiter, PhD, Department of Anatomy and Embryology, Leiden University, Wassenaarseweg 62, PO Box 9602, 2300 RC Leiden, the Netherlands. E-mail RUITER{at}RULLF2.leidenuniv.nl


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract All blood vessels are lined by endothelium and, except for the capillaries, surrounded by one or more layers of smooth muscle cells. The origin of the embryonic vascular smooth muscle cell has until now been described from neural crest and locally differentiating mesenchyme. In this study, we have substantial evidence that quail embryonic endothelial cells are competent in the dorsal aorta of the embryo to transdifferentiate into subendothelial mesenchymal cells expressing smooth muscle actins in vivo. At the onset of smooth muscle cell differentiation, QH1-positive endothelial cells were experimentally labeled with a wheat germ agglutinin–colloidal gold marker (WGA-Au). No labeled subendothelial cells were observed at this time. However, 19 hours after the endothelial cells had endocytosed, the WGA-Au–labeled subendothelial mesenchymal cells were observed in the aortic wall. Similarly, during the same time period, subendothelial cells that coexpressed the QH1 endothelial marker and a mesenchymal marker, {alpha}-smooth muscle actin, were present. In such cells, QH1 expression was reduced to a cell membrane localization. A similar antigen switch was also observed during endocardial-mesenchymal transformation in vitro. Our results are the first direct in vivo evidence that embryonic endothelial cells may transdifferentiate into candidate vascular smooth muscle cells. These data arouse new interpretations of the origin and differentiation of the cells of the vascular wall in normal and diseased vessels.


Key Words: {alpha}-smooth muscle actin • endothelial cell • embryo • smooth muscle cell • vessel wall


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Blood vessel formation in the embryo starts by differentiation of ECs from the splanchnic mesoderm, probably induced by basic fibroblast growth factor.1 By way of vasculogenesis and subsequent migration, the ECs form a vessel network.2 3 4 In general, it is thought, but not proven, that in situ the subendothelial SMC differentiates from mesenchymal cells derived, in turn, from the primitive streak.5 6 7 Cell-tracing techniques, such as chicken-quail chimeras8 and retroviral reporter gene transfer,9 10 have shown that SMCs in the head and neck region may also differentiate from neural crest cells.

A first indication of media formation is observed as mesenchymal condensations spreading around an endothelial tube.11 This process proceeds from caudal to cranial areas and is initially patchy.12 During thickening of the aortic media, the premature SMCs can be distinguished from neighboring non-SMCs by the expression of muscle-specific actins. During subsequent maturation,13 14 15 16 17 18 SMCs start to express contractile proteins, such as {alpha}-SM actin, SM myosin light and heavy chains, and cytoskeletal proteins, such as desmin, filamin, and SM-phosphoglucomutase–related protein, whereafter the regulatory proteins associated with the myosin and actin complex, such as myosin light chain kinase, tropomyosin, SM-22, h-caldesmon, and calponin, are expressed to complete the SMC phenotype.

To study the complete SMC lineage in the embryo, a marker is necessary that is present during all stages of differentiation and maturation of SMCs. Until now, none of the known "SMC-specific" proteins is restricted to SMCs and is present during the entire lifetime.15 Except for the late differentiation markers, such as myosin light chain kinase and calponin,13 the earlier SMC proteins are also (transiently) expressed by other muscle and nonmuscle cell types.14 19 20 21 Recent data on SM22 expression indicate that it is an early marker in the chick13 and mouse,17 18 but not solely SMC specific. Therefore, to study the SMC lineage, it is important to use differentiation-independent markers, such as heterospecific cells, retroviral reporter gene transfer, or wheat germ agglutinin–colloidal gold incorporation.

Arciniegas et al22 reported that adult bovine ECs can transdifferentiate into SM-like cells in vitro. They showed TGF-ß1–induced {alpha}-SM actin expression in aortic ECs, whereas expression of factor VIII–related antigen was lost. Moreover, several experiments show that embryonic endocardial (=endothelial) cells can transdifferentiate into mesenchymal cells during cushion formation.23 24 25 If already specialized ECs can interconvert into a phenotype that is more typical of SMCs in vitro, the following old question can be addressed: Do ECs and SMCs in vivo belong to distinct cell lineages, or do they convert their phenotype during developmental or pathological processes? In contrast to SMCs, the characteristic epithelial phenotype of ECs in vivo appears to be quite immutable. Several authors,26 27 28 however, have speculated that ECs can give rise to a population of subendothelial cells during atherosclerotic intimal thickening.

To study a possible endothelial origin for embryonic aortic SMCs in vivo, quail ECs were labeled with WGA-Au by injection into the lumen of the vascular system of quail embryos at stage HH14-15. At this stage, the dorsal aortas between the pharyngeal arch area and the more caudal fusion site do not as yet have cells that express SM actin. WGA-Au has been used before as a cell-lineage marker, eg, for neural crest and primitive streak.29 30 Wheat germ agglutinin binds with high affinity to N-acetylglucosamine oligomers31 that are present on the cell membrane of ECs. After endocytosis of WGA-Au, the fate of individual labeled ECs can be traced even after delamination from the endothelial monolayer. In addition, immunohistochemistry was performed to study the change in antigen expression of delaminated cells.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Animals
Fertile quail eggs (Coturnix coturnix) were incubated for 48 hours (stage HH14-15) at 37°C and 60% relative humidity. The quail embryos were staged according to the age-determination criteria of Hamburger and Hamilton.32 The egg shell was windowed, and the membranes of the air chamber were removed.

Colloidal Gold Injections
The colloidal gold solution (average particle size, 12 nm) was prepared according to Smits-van Prooije et al.29 The colloidal gold solution precipitates easily in {approx}15 minutes into large aggregates, which can no longer be endocytosed by the cells. Therefore, the colloidal gold solution needs to be freshly prepared and stored in the dark at 4°C. A small amount ({approx}10 µL) of the solution was injected into the left or right anterior vitelline vein by a glass needle (tip diameter, 10 to 15 µm) that was connected by a pressure-insensitive oil-filled tube to a Hamilton syringe, as was described previously.33 After injection, the eggs were sealed with tape and reincubated for 5 minutes, 10 minutes, 30 minutes, 5 hours, or 19 hours. Subsequently, the embryos were removed from the yolk, rinsed several times in PBS, pH 7.2, and fixed for 24 hours in half-strength Karnovsky fixative34 for TEM. For immunohistochemical analysis, the embryos were fixed for 48 hours in 2% acetic acid in absolute ethanol or for 4 hours in periodate-lysine-paraformaldehyde35 for immuno-TEM.

TEM
The embryos were rinsed in 0.1 mol/L cacodylate buffer, pH 7.2, postfixed in 1% OsO4 for 2 hours at 4°C in the same buffer. The embryos were rinsed again, dehydrated in graded ethanol, and transferred via propylene oxide (twice for 15 minutes each) and propylene oxide/Epon 812 (Merck) (1:1, twice for 30 minutes each; 1:2, twice for 30 minutes each) to Epon 812 (2 hours at room temperature and 18 hours at 60°C). Ultrathin sections were contrasted with uranyl acetate and lead citrate.36 To obtain both low-power light microscopic and electron microscopic pictures of the same colloidal gold–labeled cells, semithin sections (2 µm) were produced and stained with toluidine blue for 5 seconds at 40°C, rinsed with distilled water, and air-dried. After photography, these sections were reembedded by placing Beem capsules (Balzers, Liechtenstein) filled with Epon 812 upside down on top of the sections. After polymerization, the capsules were removed from the glass in liquid nitrogen for 10 seconds. Ultrathin sections prepared from this same region were then studied electron-microscopically.

Immuno-TEM
Quail endothelial and hematopoietic cells are recognized by the monoclonal antibody QH137 (IgG1, Hybridoma Bank). Cellular and possible extracellular antigen location in the vessel wall was determined by immunoelectron microscopy. After they were rinsed in 0.1 mol/L phosphate buffer (pH 7.3), the embryos were dehydrated in graded ethanol. They were then placed for 1 hour in ethanol/Lowicryl K4M (1:1), 1 hour in ethanol/Lowicryl K4M (1:2), and overnight in Lowicryl K4M (Bio-Rad). Polymerization was performed for 24 hours at -20°C under 254-nm UV light. The blocks were then hardened for 48 hours at room temperature. Ultrathin sections, mounted on nickel grids, were rinsed in PBS. Overnight incubation was at 4°C with QH1, 1:500 diluted in PBS containing 1% ovalbumin. After thorough washing in PBS (three times for 5 minutes each), the sections were incubated with a second antibody, rabbit anti-mouse (Dako), in the same buffer for 2 hours at room temperature. Redundant antibodies were washed away in PBS. Protein A conjugated to 10 nm gold (Dr G. Posthuma, Laboratory for Cell Biology, Utrecht, the Netherlands; 1:50 diluted in PBS with 1% ovalbumin) was added to visualize the complexes of antibodies. The sections were washed again in PBS (three times for 5 minutes) and once in 0.1 mol/L cacodylate buffer, pH 7.2. After short postfixation in 1% glutaraldehyde in 0.1 mol/L cacodylate buffer and subsequent washing in distilled water, the sections were contrasted for 5 minutes with uranyl acetate.

Immunohistochemistry on Paraffin Sections
After dehydration in graded ethanol and 100% xylene, injected and noninjected embryos were embedded in paraffin and serially sectioned transversely at 5 µm. Deparaffination, rehydration, and washing two times in PBS and once in PBS with 0.05% Tween 20 was followed by overnight incubation at room temperature with QH1 diluted in PBS (1:500) with 0.05% Tween 20 and 1% ovalbumin. After they were washed repeatedly, sections were then incubated with the second primary mouse antibody 1A4, {alpha}-SM actin specific (IgG2a, Dako; M851), in the dilution buffer (1:100) for 3 hours. The sections were incubated sequentially with secondary antibodies, goat anti-mouse IgG1-FITC (Boehringer-Mannheim; 100823) and goat-anti-mouse IgG2a-TRITC (Southern Biotechnology Associates; 1080-03), in the dilution buffer (1:100), each for 1 hour at room temperature in a dark chamber, with repeated washings in between. Negative controls were performed by omitting a primary or secondary antibody from a selected section. The muscle actin–specific antibody HHF35 (IgG1, Dako M635) was used to confirm the SMC characteristics in separate incubations at a dilution of 1:1000. Subsequently, the sections were washed again and mounted in 2% triethylenediamine (D522, Sigma Chemical Co) in 90% glycerol with 10% Tris-NaCl buffer, pH 8.0. The sections were evaluated with CLSM (MRC600, Bio-Rad) in combination with reflection contrast microscopy. The optical section thickness was 1.2 µm. FITC excitation was studied with standard K1 filters and TRITC excitation with K2 filters. To visualize the colloidal gold–positive vesicles, standard Biorad REFL and AREF filters were used. To merge the files, the various primary pictures were recorded at the same optical level.

Cell Culture
Heart tubes of quail embryos, stages HH14, 15, and 18, containing both atrioventricular and ventricular endocardium were longitudinally opened. The hearts, with the endocardium facing the bottom, were placed on 35-mm plastic dishes (Nunc well). The heart tubes were allowed to adhere to the plastic for 3 to 4 hours with a minimal of culture medium 199 (GIBCO), containing 1% chick serum (Spafas), 5 µg/mL insulin, 5 µg/mL transferrin, 5 µg/mL selenium ITS (Collaborative Research), and streptomycin/penicillin (GIBCO). After that time had elapsed, complete medium was carefully added to prevent detachment from the plastic. After it was cultured overnight, the myocardium was removed by microdissection using a 10-gauge needle. Endocardial cells formed monolayers that survived 2 or more days, depending on the developmental stage of the explanted tissue. The explants were fixed in 70% ethanol, and after rehydration, they were incubated overnight in PBS containing 1% bovine serum albumin. The cell cultures were incubated for 1 hour with both QH1 and 1A4 and subsequent incubation with secondary fluorescently labeled antibodies as essentially described above. After they were washed, the cultures were mounted in glycerol.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
EC Tracing
WGA-Au was injected into the vascular system of stage HH14-15 quail embryos before a tunica media with SMCs around the endothelium of the descending aorta had developed. Within 5 minutes after injection, many clathrin-coated pits and endocytic vesicles containing WGA-Au were found in the ECs at the TEM detection level (Fig 1ADown). Leakage of WGA-Au through endothelial fenestrae into the subendothelial space and endocytosis by neighboring mesenchymal cells was not seen in any sections examined by TEM. After 30 minutes, unbound WGA-Au was no longer detectable in the lumen of the aorta, and the coated pits in the ECs were almost absent. From this finding, we concluded that the uptake process is bound by a time span of about half an hour. The ECs contained large endocytic vesicles from that moment on (Fig 1BDown), probably as a result of fusion of numerous endocytic vesicles and primary lysosomes. Exocytosis of the WGA-Au–containing vesicles by fusion with the outer cell membrane was not observed. To exclude cell-to-cell gold-label transfer, the embryos were checked for cell death and subsequent phagocytosis of cellular remnants by neighboring mesenchymal cells. In none of the stages (5 minutes, 30 minutes, 5 hours, and 19 hours after labeling) was EC death or the presence of cell debris containing phagosomes in mesenchymal cells observed. Because the detection of degenerated cells that have been phagocytosed may remain possible up to 24 hours,38 it is unlikely that we have overlooked significant numbers of apoptotic cells. Because ectopic WGA-Au was never found in the subendothelial spaces, even after rigorous and repeated attempts to find it, we suggest that this approach for in vivo labeling is a reliable procedure for determining cell fate.



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  		Figure 1. Transmission electron micrographs of WGA-Au–labeled vacuoles in ECs and SMCs of the wall of the descending aorta of embryos that were injected with WGA-Au at stage HH14-15. The cells were first examined and localized by light microscopy. A, The WGA-Au is endocytosed by coated pits of ECs (arrowhead) and blood cells (not shown). Within 5 minutes, endocytic vesicles containing WGA-Au are present (arrow). Unbound WGA-Au is seen in the lumen (L) of the aorta, but no leakage of the WGA-Au through fenestrae is detected in the subendothelial space between the mesenchymal cells (M). B, After 30 minutes, the ECs contain larger endosomes as a result of fusion of numerous endocytic vesicles. No unbound WGA-Au or labeled mesenchymal cells are present. C, Nineteen hours after injection (stage HH20), WGA-Au–labeled mesenchymal cells subjacent to the endothelium are present. Some of these cells contain bundles of contractile filaments (asterisk), which are not present in the endothelium in any of the stages studied. d indicates the section enlarged in panel D. D, An enlargement of the outlined section of panel C is shown. Bar=0.5 µm (A, B, and D) and 5 µm (C).

At stage HH20, 19 hours after injection, both gold-labeled endothelial and subendothelial cells were detected light microscopically. After they were prepared by reembedding and ultrathin sectioning, the same subendothelial cells could be studied by electron microscopy. The WGA-Au in the subendothelial cells was present in large endosomes (Fig 1CUp and 1DUp). The first cells were detected at the ventral and dorsal sides of the aorta. Some of these cells still resembled ECs, containing many mitochondria and a large amount of rough endoplasmic reticulum, whereas others already had a contractile apparatus with fewer mitochondria and less rough endoplasmic reticulum (Fig 1DUp). Thirty minutes after injection, virtually all ECs were labeled. Thereafter, the combined effects of cell division and asymmetric distribution of the gold label precluded further attempts to clearly determine the incidence of labeled ECs versus mesenchymal cells.

Differentiation Markers
The ultrastructural and WGA-Au data were confirmed by an immunohistochemical approach using differentiation markers. ECs of the quail dorsal aorta were characterized by the endothelium-specific monoclonal antibody QH1. The QH1 antigen is detected in the TEM on both the luminal and abluminal membranes of ECs (Fig 2ADown). Moreover, all ECs contain cytoplasmic aggregations of the antigen. Most likely, this cytoplasmic expression of the protein is restricted to the Golgi apparatus or vesicles. Prospective SMCs were defined by the presence of actin filaments, recognized by HHF35 monoclonal antibody (specific for muscle actins in general) and 1A4 (specific for {alpha}-SM actin). These actin stainings can be considered to represent the first differentiation markers of prospective SMCs.



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  		Figure 2. Immunoelectron micrographs of the aortic wall incubated with QH1. A, At all stages of development, the QH1 epitope (arrows) is detectable in ECs in both the (ab)luminal cell membranes and cytosol. Cytoplasmic expression is restricted to small areas in the EC, indicating that we are dealing with vesicles or Golgi apparatus. B, At those places of EC-mesenchymal transformation (stage HH18), the antigen is still expressed in the membranes and cytosol of the ECs. However, the QH1 antigen is also expressed in the membranes of subendothelial cells, but not in the cytosol. The membrane expression of the subendothelial cells is more patchy than that of ECs. In some of the subendothelial mesenchymal cells (M), bundles of contractile filaments (asterisk) are visible. Moreover, some expression is seen in the extracellular space between the mesenchymal cells (arrowheads), indicating that the antigen is probably deposited into the extracellular space. The negative nuclei show that no background staining is present. L indicates lumen. Bar=5 µm.

Fig 3Down shows representative CLSM fluorescence micrographs in combination with CLSM–reflection contrast microscopy to detect the fluorescence at the same optical level as the vesicles containing WGA-Au. At stage HH18, some of the subendothelial cells contained gold particles, which were incorporated at stage HH15, when they lined the lumen of the descending aorta as ECs. Because they are characterized by the presence of actin filaments, they are regarded as candidate SMCs. ECs lining the lumen do not express {alpha}-SM actin at any stage.



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  		Figure 3. Descending aorta at stage HH18 of an embryo that was injected with WGA-Au at stage HH15 to label the ECs. A, CLSM fluorescence image of QH1 (green) and 1A4 (red) expression. The ECs are QH1 positive but do not express {alpha}-SM actins. The mesenchymal cells (SMCs) subjacent to the endothelium express {alpha}-SM actins. B, Confocal image of 1A4 expression at the same optical level as panel A in combination with reflection contrast microscopy to visualize WGA-Au (yellow), which has been endocytosed by the ECs at stage HH15. The candidate SMCs have endosomes containing WGA-Au, showing that they originate from EC labeled at stage HH15. During their transformation into candidate SMCs, the ECs lose their QH1 expression, while they start to produce bundles of actin filaments. L indicates lumen of the descending aorta. Bar=5 µm.

As shown with TEM, the QH1 antibody recognizes both cytoplasmic and cell membrane–bound epitopes. Until stage HH13, the QH1 staining was confined to ECs lining the lumen. Subsequently, at stage HH16, just before actin expression, in the mesenchyme surround-ing the aorta, the QH1 antigen is also expressed and visualized as thin lines in the CLSM (Fig 4ADown). The extraendothelial QH1 expression is mainly localized to the cell surface membranes of the stained mesenchymal cells (Fig 2BUp), but expression is not as abundant as in the ECs; expression in the cytosol was not detected. At stage HH18, many of these subendothelial mesenchymal cells, showing extensive actin filament bundles (Fig 2BUp and 4BDown), still express QH1 in their cell membrane (Fig 4BDown). In later stages, all cells in the condensed layer around the aorta are actin positive but QH1 negative.



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  		Figure 4. CLSM fluorescence images of QH1 (green) and 1A4 (red) expression in the descending aorta of a noninjected embryo. A, At stage HH16, ECs at the dorsal side (D) of the descending aorta delaminate from the endothelial monolayer into surrounding mesenchyme. The QH1 epitope is still present in parts of the cell membrane of mesenchymal cells (arrows), which is consistent with an endothelial origin. Ventrally (V) delaminated ECs are already differentiating into prospective SMCs as they express 1A4. B, At stage HH18, the number of mesenchymal cells that express {alpha}-SM actin has increased, although mesenchymal QH1 expression is still present. The yellow color is double expression of QH1 and 1A4 or eventual slight overprojection within the optical confocal section. L indicates lumen of the descending aorta. Bar=5 µm.

Endothelial-Mesenchymal Transformation In Vitro
The shift in antigen expression was also studied in vitro, using stage HH15-18 heart explants of ventricular and atrioventricular endocardial cells. During the culture period, endocardial cells from the ventricular region did not transform into mesenchyme and did not lose QH1 expression or acquire {alpha}-SM actin. In contrast, the atrioventricular endocardial cells demonstrated within 2 days of culture a gradual loss of their epithelium-like morphology as they migrated peripherally and acquired mesenchymal characteristics (Fig 5Down). The abundant cell-cell contacts and QH1 expression observed in cells remaining in the center of the explant (Fig 5ADown) progressively disappeared in cells located at the periphery. Cells at the periphery were more separated and spread out. This change in morphology was accompanied by a transition in protein expression. The initial endothelium-specific QH1 expression decreased, whereas {alpha}-SM actin expression increased. The presence of transitional cells, expressing both QH1 and {alpha}-SM actin, indicates that we are dealing with a gradual transformation process.



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  		Figure 5. Double staining of QH1 and {alpha}-SM actin during endothelial-mesenchymal transformation in the atrioventricular canal in vitro. A, Center of the endocardial explant after 2 days of incubation on plastic. The ECs have maintained their epithelium-like phenotype. All the ECs express the QH1 epitope (green), especially in the areas of the cell borders. There is no {alpha}-SM actin staining. B, Peripheral zone of the same endocardial explant. Most cells have lost the pattern of an epithelium-like mosaic. Extensive cell-cell separation and cell scattering are observed for the endocardium-derived mesenchymal-like cells. Some of these cells express both markers (yellow), although most cells already predominantly express {alpha}-SM actin (red). Bar=50 µm.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
We suggest that the most likely interpretation of the WGA-Au–labeling data and the double immunohistochemical staining is that ECs delaminate from the endothelial monolayer during embryonic media formation. The ECs appear to migrate into the subendothelial space, where they lose the quail endothelium-specific marker and start to express {alpha}-SM actin. Significantly, a similar mechanism of loss of the QH1 expression pattern and subsequent appearance of {alpha}-SM actin were also shown with the endocardial explant technique, which is an appropriate model for the endocardial-mesenchymal transformation during cushion formation in the heart.39 40

The WGA-Au–labeling technique has been described as a reliable method for cell-lineage studies of the neural crest and primitive streak.29 30 In the present study, the injection of WGA-Au into the vascular system similarly indicates a lineage for candidate SMC precursor cells from the endothelium of the dorsal aorta at the lower thoracic level. Leakage of WGA-Au through fenestrae was not detected, nor was phagocytosis of labeled ECs by neighboring mesenchymal cells observed. Therefore, the WGA-labeling technique was also considered to be a reliable method to study the cell lineage of the EC. Because no differences in antigen expression (QH1 and 1A4) between labeled and nonlabeled embryos were found, there is no indication that the transformation of ECs into mesenchymal cells was induced by the lectin.

QH137 as a marker for ECs has been accepted for many studies on vasculogenesis. It has a disadvantage, however, because it is also known to label hematopoietic precursors in the para-aortic mesenchyme surrounding the dorsal aorta.37 We have also observed this phenomenon in our material, but limited to the lateroventral aortic wall containing large globular cells.41 The difference with our cells is that they are double labeled with QH1 and {alpha}-SM actin and are clearly circularly positioned around the vessel.

A likely candidate to regulate EC-SMC transformation is the ES130 protein, which plays an essential role in endocardial-mesenchymal transformation during cushion formation in the heart.24 Preliminary data of ES130 protein expression in the descending aorta (HH16) show that aortic ECs also express the ES130 protein in the area in which we observe transformation into mesenchymal cells. The presence of ES130 in vascular endothelium is consistent with its proposed role as an inducer of cushion mesenchyme formation. Likewise, TGF-ß1 is a candidate for regulation or initiation of transformation, because it has been shown to promote differentiation of ECs into SM-like cells in vitro.22 To be clear, we propose ECs as but one potential source of mesenchymal cells, particularly those that would lie closest to the endothelium and, by dint of their position, could reasonably be expected to become part of the intima. If this potential subpopulation of mesenchyme is formed in a manner analogous to cushion mesenchyme, these potential vascularly derived ECs might be anticipated to also express markers similar to atrioventricularly derived mesenchyme besides {alpha}-SM actin. Studies are in progress to determine if such markers, eg, JB3,25 fibulin,42 and ES130,24 are correlatively expressed.

Thus, we believe our data are the first direct in vivo evidence that embryonic endothelium may give origin to mesenchymal cells that express {alpha}-SM actin. The capacity for ECs to be able to start expressing {alpha}-SM actin is supported by the in vitro endothelial-mesenchymal transformation data of adult aortic ECs.21

Because a high rate of cell division occurs during the embryonic stages, studied application of the colloidal gold technique was limited to {approx}24 hours or less after administration of the label. When the embryos survive until stage HH18, {alpha}-SM actin expression in the mesenchyme is one of the first indications of vessel wall differentiation. However, {alpha}-SM actin expression is not restricted to medial SMCs, is reported in a number of adult cell types, such as myofibroblasts,43 tumors,44 and ECs,22 and is transiently expressed in various embryonic tissues, such as the myotomes of the somites and myocardium.45 During endocardial-mesenchymal transformation in the atrioventricular cushions, {alpha}-SM actin is also transiently expressed in vivo.46 Actin is necessary for cell motility and migration. Recent theories of cell locomotion are based on cycles of attachment, proteolysis, contraction, and detachment, which are critical steps for an invasive behavior of cells.47 Thus, it is likely that the expression of {alpha}-SM actin is functionally related to the invasive behavior of endothelium-derived mesenchymal cells, as can be observed in the endocardial cushions. From the literature,12 48 49 however, it is evident that the expression of {alpha}-SM actin in the mesenchymal cells around the aorta is not transient and will gradually be strengthened with other (SMC-specific) proteins, such as myosin light chain kinase, calponin,13 1E12,50 and SM-22.17 18 Expression patterns of 1E1250 and SM-2213 in the descending aorta recapitulate the pattern of our {alpha}-SM actin, showing that we are dealing with differentiating SMCs. This indicates that although {alpha}-SM actin expression is not limited to SMCs in general, it is a valuable marker to study the earliest vessel wall formation.

The present study was performed at a level of the dorsal aorta just below the pharyngeal arch area, where we know that neural crest cells do not contribute to the SMC population of the vessel wall.51 We performed the study above the site of fusion of the dorsal aortas because the fusion site is complicated and mechanistically not well understood. Additional experiments will be needed to substantiate whether the EC-mesenchymal transformation is a generalized process that occurs at every site of vessel formation in the embryo or whether it occurs only at certain sites. The presence of von Willebrand factor, which is an EC-specific protein, in subendothelial cells of the intimal cushion of a normal closing ductus arteriosus52 suggests that the capacity of ECs to transform is not restricted to embryonic development. This idea is additionally supported by the observation of QH1-positive subendothelial intimal cells in atherosclerotic plaques of the quail.53

Literature data refer to endothelium-derived cells containing SM actin in various pathological processes, such as restenosis,26 inflammation, and hypertension,27 and to their induction in in vitro experiments.22 The impact of our findings for both development and disease of the vessel wall needs further study. SMC heterogeneity54 55 56 is already a well-described phenomenon in fetal, neonatal, and adult vessel wall, and its impact for disease processes forms the basis of many studies. If, however, in fully differentiated stages in the adult the EC can participate in the formation of intimal thickening, this might open new ways of thinking in controlling this process in the setting of endothelium-mediated gene therapy.


*    Selected Abbreviations and Acronyms
 
CLSM = confocal laser scanning microscopy
EC = endothelial cell
HH = Hamburger-Hamilton developmental stage
(associated with number)
SM = smooth muscle
SMC = smooth muscle cell
TEM = transmission electron microscopy
TGF = transforming growth factor
WGA-Au = wheat germ agglutinin–colloidal gold marker


*    Acknowledgments
 
M.M.T. Mentink and B.C.M. Vrolijk are gratefully acknowledged for their skillful technical assistance.

Received September 6, 1996; accepted December 19, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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